WO2014090583A1 - Delphinidin for combating melanoma cells - Google Patents
Delphinidin for combating melanoma cells Download PDFInfo
- Publication number
- WO2014090583A1 WO2014090583A1 PCT/EP2013/074957 EP2013074957W WO2014090583A1 WO 2014090583 A1 WO2014090583 A1 WO 2014090583A1 EP 2013074957 W EP2013074957 W EP 2013074957W WO 2014090583 A1 WO2014090583 A1 WO 2014090583A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- delphinidin
- composition
- cells
- use according
- sbe
- Prior art date
Links
- GCPYCNBGGPHOBD-UHFFFAOYSA-N Delphinidin Natural products OC1=Cc2c(O)cc(O)cc2OC1=C3C=C(O)C(=O)C(=C3)O GCPYCNBGGPHOBD-UHFFFAOYSA-N 0.000 title claims abstract description 76
- 235000007242 delphinidin Nutrition 0.000 title claims abstract description 72
- 201000001441 melanoma Diseases 0.000 title claims abstract description 24
- JKHRCGUTYDNCLE-UHFFFAOYSA-O delphinidin Chemical compound [O+]=1C2=CC(O)=CC(O)=C2C=C(O)C=1C1=CC(O)=C(O)C(O)=C1 JKHRCGUTYDNCLE-UHFFFAOYSA-O 0.000 title claims abstract 6
- 239000000203 mixture Substances 0.000 claims abstract description 50
- 229920000858 Cyclodextrin Polymers 0.000 claims abstract description 28
- 238000011282 treatment Methods 0.000 claims abstract description 22
- 229960004853 betadex Drugs 0.000 claims abstract description 18
- 239000001116 FEMA 4028 Substances 0.000 claims abstract description 16
- 208000035250 cutaneous malignant susceptibility to 1 melanoma Diseases 0.000 claims abstract description 15
- 125000004964 sulfoalkyl group Chemical group 0.000 claims abstract description 13
- 239000000243 solution Substances 0.000 claims description 34
- 206010028980 Neoplasm Diseases 0.000 claims description 20
- 230000006907 apoptotic process Effects 0.000 claims description 16
- 239000013543 active substance Substances 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 108010050904 Interferons Proteins 0.000 claims description 8
- 102000014150 Interferons Human genes 0.000 claims description 8
- 229960005486 vaccine Drugs 0.000 claims description 8
- 229940047124 interferons Drugs 0.000 claims description 7
- 238000009472 formulation Methods 0.000 claims description 6
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical group O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 238000002512 chemotherapy Methods 0.000 claims description 5
- 239000000824 cytostatic agent Substances 0.000 claims description 5
- 238000001959 radiotherapy Methods 0.000 claims description 5
- 108010047761 Interferon-alpha Proteins 0.000 claims description 4
- 102000006992 Interferon-alpha Human genes 0.000 claims description 4
- 230000001085 cytostatic effect Effects 0.000 claims description 4
- 238000009169 immunotherapy Methods 0.000 claims description 4
- 239000003446 ligand Substances 0.000 claims description 4
- 238000006467 substitution reaction Methods 0.000 claims description 4
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 3
- 125000001033 ether group Chemical group 0.000 claims description 3
- 238000007918 intramuscular administration Methods 0.000 claims description 3
- 238000007912 intraperitoneal administration Methods 0.000 claims description 3
- 238000001990 intravenous administration Methods 0.000 claims description 3
- 230000002685 pulmonary effect Effects 0.000 claims description 3
- 238000007920 subcutaneous administration Methods 0.000 claims description 3
- 102000003390 tumor necrosis factor Human genes 0.000 claims description 3
- 239000000443 aerosol Substances 0.000 claims description 2
- 239000002775 capsule Substances 0.000 claims description 2
- 239000006071 cream Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000000499 gel Substances 0.000 claims description 2
- 239000006210 lotion Substances 0.000 claims description 2
- 239000002674 ointment Substances 0.000 claims description 2
- 239000006072 paste Substances 0.000 claims description 2
- 239000003381 stabilizer Substances 0.000 claims description 2
- 229940097346 sulfobutylether-beta-cyclodextrin Drugs 0.000 claims description 2
- 239000003826 tablet Substances 0.000 claims description 2
- 108700012411 TNFSF10 Proteins 0.000 claims 2
- 239000000654 additive Substances 0.000 claims 1
- 239000000945 filler Substances 0.000 claims 1
- 239000003205 fragrance Substances 0.000 claims 1
- 229940079322 interferon Drugs 0.000 claims 1
- 238000001356 surgical procedure Methods 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 189
- FFNDMZIBVDSQFI-UHFFFAOYSA-N delphinidin chloride Chemical compound [Cl-].[O+]=1C2=CC(O)=CC(O)=C2C=C(O)C=1C1=CC(O)=C(O)C(O)=C1 FFNDMZIBVDSQFI-UHFFFAOYSA-N 0.000 description 80
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 27
- 230000000694 effects Effects 0.000 description 20
- 238000000034 method Methods 0.000 description 20
- 239000003814 drug Substances 0.000 description 19
- 239000007864 aqueous solution Substances 0.000 description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Natural products OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 17
- 230000004663 cell proliferation Effects 0.000 description 16
- 229940079593 drug Drugs 0.000 description 15
- 239000000126 substance Substances 0.000 description 13
- 210000004881 tumor cell Anatomy 0.000 description 13
- 238000003556 assay Methods 0.000 description 12
- 238000012360 testing method Methods 0.000 description 12
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 11
- 210000002950 fibroblast Anatomy 0.000 description 11
- 239000007787 solid Substances 0.000 description 11
- 238000010790 dilution Methods 0.000 description 10
- 239000012895 dilution Substances 0.000 description 10
- 238000012544 monitoring process Methods 0.000 description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 9
- 201000011510 cancer Diseases 0.000 description 9
- NBBJYMSMWIIQGU-UHFFFAOYSA-N Propionic aldehyde Chemical compound CCC=O NBBJYMSMWIIQGU-UHFFFAOYSA-N 0.000 description 8
- 239000004480 active ingredient Substances 0.000 description 8
- 229930014669 anthocyanidin Natural products 0.000 description 8
- 235000008758 anthocyanidins Nutrition 0.000 description 8
- 210000002889 endothelial cell Anatomy 0.000 description 8
- 239000006285 cell suspension Substances 0.000 description 7
- 229940097362 cyclodextrins Drugs 0.000 description 7
- 230000006698 induction Effects 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 230000001640 apoptogenic effect Effects 0.000 description 6
- 239000012482 calibration solution Substances 0.000 description 6
- 230000003833 cell viability Effects 0.000 description 6
- 230000006882 induction of apoptosis Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- JUJBNYBVVQSIOU-UHFFFAOYSA-M sodium;4-[2-(4-iodophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate Chemical compound [Na+].C1=CC([N+](=O)[O-])=CC=C1N1[N+](C=2C=CC(I)=CC=2)=NC(C=2C(=CC(=CC=2)S([O-])(=O)=O)S([O-])(=O)=O)=N1 JUJBNYBVVQSIOU-UHFFFAOYSA-M 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 150000001452 anthocyanidin derivatives Chemical class 0.000 description 5
- 230000001028 anti-proliverative effect Effects 0.000 description 5
- 235000011175 beta-cyclodextrine Nutrition 0.000 description 5
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 238000007422 luminescence assay Methods 0.000 description 5
- -1 melphatan Chemical compound 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- ZTQSAGDEMFDKMZ-UHFFFAOYSA-N butyric aldehyde Natural products CCCC=O ZTQSAGDEMFDKMZ-UHFFFAOYSA-N 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000010668 complexation reaction Methods 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 230000007123 defense Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- VNDHXHMRJVTMTK-WZVRVNPQSA-H hexasodium 4-[[(1S,3R,5R,6S,8R,10R,11S,13R,15R,16S,18R,20R,21S,23R,25R,26S,28R,30R,31S,33R,35R,36R,37R,38R,39R,40R,41R,42R,43R,44R,45R,46R,47R,48R,49R)-36,37,38,39,40,41,42,43,44,45,46,47,48,49-tetradecahydroxy-10-(hydroxymethyl)-15,20,25,30,35-pentakis(4-sulfonatobutoxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29,32,34-tetradecaoxaoctacyclo[31.2.2.23,6.28,11.213,16.218,21.223,26.228,31]nonatetracontan-5-yl]methoxy]butane-1-sulfonate Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].OC[C@H]1O[C@@H]2O[C@H]3[C@H](O)[C@@H](O)[C@H](O[C@@H]3COCCCCS([O-])(=O)=O)O[C@H]3[C@H](O)[C@@H](O)[C@H](O[C@@H]3COCCCCS([O-])(=O)=O)O[C@H]3[C@H](O)[C@@H](O)[C@H](O[C@@H]3COCCCCS([O-])(=O)=O)O[C@H]3[C@H](O)[C@@H](O)[C@H](O[C@@H]3COCCCCS([O-])(=O)=O)O[C@H]3[C@H](O)[C@@H](O)[C@H](O[C@@H]3COCCCCS([O-])(=O)=O)O[C@H]3[C@H](O)[C@@H](O)[C@H](O[C@@H]3COCCCCS([O-])(=O)=O)O[C@H]1[C@H](O)[C@H]2O VNDHXHMRJVTMTK-WZVRVNPQSA-H 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 210000003491 skin Anatomy 0.000 description 4
- 239000008107 starch Substances 0.000 description 4
- 235000019698 starch Nutrition 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- ODLHGICHYURWBS-LKONHMLTSA-N trappsol cyclo Chemical compound CC(O)COC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)COCC(O)C)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1COCC(C)O ODLHGICHYURWBS-LKONHMLTSA-N 0.000 description 4
- 206010027476 Metastases Diseases 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000000875 corresponding effect Effects 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 231100000673 dose–response relationship Toxicity 0.000 description 3
- NWKFECICNXDNOQ-UHFFFAOYSA-N flavylium Chemical compound C1=CC=CC=C1C1=CC=C(C=CC=C2)C2=[O+]1 NWKFECICNXDNOQ-UHFFFAOYSA-N 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 108091006112 ATPases Proteins 0.000 description 2
- 102000057290 Adenosine Triphosphatases Human genes 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- GXJABQQUPOEUTA-RDJZCZTQSA-N bortezomib Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)B(O)O)NC(=O)C=1N=CC=NC=1)C1=CC=CC=C1 GXJABQQUPOEUTA-RDJZCZTQSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 229940127089 cytotoxic agent Drugs 0.000 description 2
- 231100000599 cytotoxic agent Toxicity 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 210000004925 microvascular endothelial cell Anatomy 0.000 description 2
- 210000004088 microvessel Anatomy 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 229960002378 oftasceine Drugs 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 239000012088 reference solution Substances 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000009097 single-agent therapy Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- 125000003831 tetrazolyl group Chemical group 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- UEJJHQNACJXSKW-UHFFFAOYSA-N 2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3(2H)-dione Chemical compound O=C1C2=CC=CC=C2C(=O)N1C1CCC(=O)NC1=O UEJJHQNACJXSKW-UHFFFAOYSA-N 0.000 description 1
- YXFNFSBQEDFMHR-UHFFFAOYSA-N 2-(2-sulfoethoxy)ethanesulfonic acid Chemical compound OS(=O)(=O)CCOCCS(O)(=O)=O YXFNFSBQEDFMHR-UHFFFAOYSA-N 0.000 description 1
- 238000009636 ATP test Methods 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- DLGOEMSEDOSKAD-UHFFFAOYSA-N Carmustine Chemical compound ClCCNC(=O)N(N=O)CCCl DLGOEMSEDOSKAD-UHFFFAOYSA-N 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 230000010337 G2 phase Effects 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 102100040018 Interferon alpha-2 Human genes 0.000 description 1
- 102100026720 Interferon beta Human genes 0.000 description 1
- 108010079944 Interferon-alpha2b Proteins 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- 229910010199 LiAl Inorganic materials 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000037602 Platelet Endothelial Cell Adhesion Molecule-1 Human genes 0.000 description 1
- 108010069381 Platelet Endothelial Cell Adhesion Molecule-1 Proteins 0.000 description 1
- 108010090931 Proto-Oncogene Proteins c-bcl-2 Proteins 0.000 description 1
- 102000013535 Proto-Oncogene Proteins c-bcl-2 Human genes 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 241000405217 Viola <butterfly> Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000746 body region Anatomy 0.000 description 1
- 229960001467 bortezomib Drugs 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- OQUUTERJWTYTHP-UHFFFAOYSA-N butanedioate;1h-tetrazol-1-ium Chemical compound [NH2+]1C=NN=N1.[NH2+]1C=NN=N1.[O-]C(=O)CCC([O-])=O OQUUTERJWTYTHP-UHFFFAOYSA-N 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- BQRGNLJZBFXNCZ-UHFFFAOYSA-N calcein am Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O)=C(OC(C)=O)C=C1OC1=C2C=C(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(=O)C)C(OC(C)=O)=C1 BQRGNLJZBFXNCZ-UHFFFAOYSA-N 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 229960005243 carmustine Drugs 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 210000001608 connective tissue cell Anatomy 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 229960000684 cytarabine Drugs 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 239000002254 cytotoxic agent Substances 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 229960003957 dexamethasone Drugs 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 230000005670 electromagnetic radiation Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- LIYGYAHYXQDGEP-UHFFFAOYSA-N firefly oxyluciferin Natural products Oc1csc(n1)-c1nc2ccc(O)cc2s1 LIYGYAHYXQDGEP-UHFFFAOYSA-N 0.000 description 1
- 229940080345 gamma-cyclodextrin Drugs 0.000 description 1
- GDSRMADSINPKSL-HSEONFRVSA-N gamma-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO GDSRMADSINPKSL-HSEONFRVSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000009830 intercalation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- 210000002752 melanocyte Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 238000004377 microelectronic Methods 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Polymers 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- JJVOROULKOMTKG-UHFFFAOYSA-N oxidized Photinus luciferin Chemical compound S1C2=CC(O)=CC=C2N=C1C1=NC(=O)CS1 JJVOROULKOMTKG-UHFFFAOYSA-N 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 210000004694 pigment cell Anatomy 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 229960004618 prednisone Drugs 0.000 description 1
- XOFYZVNMUHMLCC-ZPOLXVRWSA-N prednisone Chemical compound O=C1C=C[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 XOFYZVNMUHMLCC-ZPOLXVRWSA-N 0.000 description 1
- 230000000861 pro-apoptotic effect Effects 0.000 description 1
- 230000005522 programmed cell death Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 238000012342 propidium iodide staining Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000012217 radiopharmaceutical Substances 0.000 description 1
- 229940121896 radiopharmaceutical Drugs 0.000 description 1
- 230000002799 radiopharmaceutical effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 230000035806 respiratory chain Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 229960003433 thalidomide Drugs 0.000 description 1
- 210000002105 tongue Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229940099039 velcade Drugs 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000003026 viability measurement method Methods 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
- A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
- A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Definitions
- compositions comprising
- malignant melanoma also known as "black skin cancer” known, it is a wicked ⁇ -like transformation of the pigment cells, called melanocytes.
- melanocytes The cancer already inclined at an early stage for spreading metastases through blood and lymph vessels.
- Malignant melanoma is curable, at least if it is diagnosed and treated early.
- the main method of treatment is surgical Ent ⁇ fernung the tumor alongside radiotherapy, although monotherapy with interferons in the treatment of malignant melanoma applies.
- vaccine therapy ie the active immunotherapy with ⁇ means tumor vaccines using tumor vaccines to stimulate the body's defense against targeted control of cancer cells.
- the immune cells are characterized by the administered vaccine features of the tumor, (for example, protein molecules that are produced by the tumor cells or cell fragments of tumor cells) presented so that the immune cells recognize these features as foreign and kör ⁇ pereigene cells that carry these features attack .
- chemotherapy ie drug treatment with chemical substances that damage or inhibit tumor cells (cytostatics), is also of practical relevance.
- the object of the present invention is to provide an effective means as an alternative or supplement to treatment options of the malignant melanoma known from the prior art.
- the composition of the invention is used to treat an object or individual suffering from malignant melanoma.
- object includes live animals and humans
- the purpose of this treatment is the at least partial killing or neutralization of the tumor cells.
- Negtralization and “killing” in the sense of the present invention mean the at least partial destruction or dissolution or inactivation or multiplication of the tumor cells .
- the present invention also relates to methods of treating a patient suffering from malignant melanoma object, wherein the object comprises a therapeutically effective amount of he ⁇ inventive composition is administered.
- the object comprises a therapeutically effective amount of he ⁇ inventive composition is administered.
- Melanomzel- len fight with the drug but then possible to have too much as the vitality and proliferation of non ⁇ cancer cells unaffected.
- treatment means the whole or part of reaching the nachfol ⁇ quietly mentioned results: the whole or partial reduction of the disease, improvement of at least one of the clinical symptoms or associated with the disease indicators, delay, suppression or protection against Disease progression, or partial or total delay, suppression or protection against the onset or onset of the disease
- the object to be treated is a human or animal, preferably a mammal
- Veterinary treatment includes treatment of beneficial or wild animals (eg sheep , cats, horses, cows, pigs), laboratory animals (eg rats, mice, guinea pig ⁇ ne, monkeys).
- composition comprising a complex of delphinidin and a sulfoalkyl ether-.beta.-cyclodextrin and / or delphinidin or its salts
- composition includes the composition as a single preparation, ie without further therapeutically ⁇ schematically active components.
- the composition may be at least one other therapeutically active Sub ⁇ substance include.
- the composition of the invention may be used alone or in combination with at least one other therapeutic agent for the reduction of one or several symptoms of malignant melanoma.
- Administration of the composition of the invention may be administered concomitantly with the other therapeutic substance, which may be part of the same composition or provided in a different composition.
- the composition of the invention can be administered before or after administration of the other therapeutic substance.
- the erfindungsge ⁇ Permitted composition may be administered via the same or a different route of administration, we the other therapeutic Sub ⁇ substance.
- the other therapeutically active substance is vorzugswei ⁇ se a tumor necrosis factor-related apoptosis-induz FOURTH ligand (Tumor Necrosis Factor Related Apoptosis I_nducing Ligand - TRAIL).
- the further therapeutically effective substance is selected from the group consisting of Zy ⁇ tostatika, interferons, preferably alpha and / or beta interferons, more preferably interferon alpha 2-a and alpha 2-b, and tumor vaccines , The latter can also be combined in combi ⁇ nation with TRAIL and applied.
- a combination of chemotherapy and immunotherapy, and radiation therapy may be appropriate to achieve a ⁇ of individual metastases by regressions.
- interferons in particular of the white blood cells (leucocytes), IFN-alpha, and connective tissue cells are commonly used.
- Fibroblasts formed IFN-beta, using the Inhibit growth of healthy as well as malignant cells and stimulate the immune system.
- übli ⁇ chlay use is made of pure, genetically derived Interfe ⁇ ron, for example, on the approved in Germany for cancer treatment interferon alpha 2-a (Rofe- ron®) and interferon alpha 2-b (IntronA®).
- the original concept in which the body's own tumor cells are rendered unable to divide by radiation and to increase the stimulus for the immune system, preferably mixed with a virus is injected into the skin to attract defense cells and targeted to activate these tumor cells, increasingly rare for use.
- protein molecules produced by the tumor cells are now regularly used, with which cells grown from blood precursor cells are loaded in vitro and thus returned to the patient to present them to the defense cells as something to be controlled.
- the same result is obtained when the gene of a blocking or activating substance for defense cells is introduced into the tumor cells.
- chemotherapeutic agents are be ⁇ features commonly called “cytostatic agents”. Cytostatics produced synthetically or Derivatives of naturally occurring cytotoxins which trigger the programmed cell death (apoptosis) of the tumor cells
- chemotherapeutic agents which are used in the context of the present invention include bortezomib (Velcade®, Millennium), melphatan, prednisone, vincristine, carmustine, cyclophosphamide, dexamethasone, thalidomide, doxorubicin, cisplatin, estrogen and cytarabine.
- radiotherapy can additionally be used.
- the radiotherapy is used preferably localized before ⁇ . That is, rays which preferably before ⁇ electromagnetic radiation and particles ⁇ in this case act locally in the irradiated area and damage the tumor cells, in particular the DNA in the nucleus by ionization and Entste ⁇ hung free radicals and breaks in the DNA of tumor cells. can be used to protect the tumor surrounding healthy tissue aperture.
- the administration of the active compound according to the invention by means of monotherapy or in combination with TRAIL or other or further therapeutic active ingredients, for example selected from the group consisting of cytostatics, interferons and tumor vaccines, can also be carried out as a so-called re ⁇ gionale therapy, for example by targeted injection (For example by means of catheter) in body regions, body cavities or in the blood vessels of the tumor region or the organ in which the tumor sits.
- a flow through the affected area or the organ is also in the way of a regional perfusion possible in which the drug flows through the area (eg arm or leg) or the organ under completion of the rest of the circuit and directly kill ⁇ leads is passed into the rest of the body to get.
- the present composition is preferably provided as a pharmaceutical composition and verab ⁇ ranges.
- pharmaceutical composition includes fully one or more active ingredients and one or more domestic nerte ingredients which act as carriers for the active ingredient or active ingredients.
- the pharmaceutical Caribbeanset ⁇ tongues allow orally administered to the complex of the invention or the composition according to the invention Rectal, parenteral, including intraperitoneal, percutaneous, subcutaneous, intramuscular, intravenous, ophthalmic, pulmonary or nasal administration
- a parenteral delivery form may be, for example, a tablet, capsule, solution, suspension or dispersion
- An ophthalmic, pulmonary or nasal dosage form may, for example an aerosol, solution, cream, paste, lotion, gel, ointment, suspension or dispersion ..
- compositions of the invention can be administered to a subject intravenously by means of a pharmaceutically acceptable carrier (eg, physiological saline).
- a pharmaceutically acceptable carrier eg, physiological saline
- physiologically acceptable buffers eg Hanks solution, Ringer's solution or physiologically buffered saline solution
- parenteral administration including intravenous, subcutaneous, intramuscular and intraperitoneal administration, an aqueous or oily solution or solid formulation is also contemplated.
- the proportion of the active ingredient in the pharmaceutical ⁇ ceutical composition may vary and is usually between 2 and 60 wt .-% of the dosage unit. The proportion of active ingredient is selected accordingly, that an effective dose is achieved.
- the delphinidin or its salts and / or complex of delphinidin and the sulfoalkyl ether-beta-cyclodextrin in a pharmaceutical preparation for the controlled and / or delayed release of the delphinidin be used.
- Salt or “pharmaceutically acceptable salt” means any pharmaceutically acceptable salt of a compound of the present invention which can release the pharmaceutically active agent or its active metabolite after administration. Salts of the compositions and complexes of the present invention may be derived from inorganic or organic acids and bases.
- the anthocyanidin delphinidin can be used in "pure” or “purified”, meaning that unwanted components have been removed.
- Cyclodextrins that can be complexed according to the invention with the anthocyanidin, delphinidin, are cyclic oligo saccharide ⁇ from CC-1, 4-glycosidically linked Glukosemole ⁇ cules.
- ß-Cyclodextrin has seven glucose units. In a sulfoalkyl ether- ⁇ -cyclodextrin, hydroxy groups of the glucose unit are etherified in a sulfoalkyl alcohol.
- Sulfoalkyl ethers are used in cyclodextrins in the prior art to increase the hydrophilicity or water solubility.
- Sulfoalkyl ether groups contribute in particular to increase the stability of the complex of anthocyanidins and correspondingly substituted .beta.-cyclodextrin, thus substantially improving the storage stability and formability of the particularly oxidation-sensitive anthocyanidins.
- the Invent ⁇ modern complex can be formulated as a storage-stable aqueous solution or solid, will be shown in more detail below.
- Particularly preferred according to the invention is the complexation of the active substance delphinidin with a sulfoalkyl ether-.beta.-cyclodextrin, preferably a sulfobutyl ether-.beta.-cyclodextrin (SBE-.beta.-CD) or a sulfoethyl ether-.beta.-cyclodextrin, which surprisingly increases the solubility and stability of the active ingredient.
- a sulfoalkyl ether-.beta.-cyclodextrin preferably a sulfobutyl ether-.beta.-cyclodextrin (SBE-.beta.-CD) or a sulfoethyl ether-.beta.-cyclodextrin
- Cyclodextrin exactly the opposite is the case, ie, the complex of active ingredient and sulfobotyl ether-ß-cyclodextrin has a significantly lower solubility compared to Kom ⁇ plex with ß-cyclodextrin result.
- the degree of substitution of the cyclodextrin with sulfoalkyl ether groups is preferably 3 to 8, more preferably 4 to 8, more preferably 5 to 8, further preferably 6 to 7.
- Suitable sulfobutyl ether- ⁇ -cyclodextrins with an average degree of substitution of 6 to 7 are described, for example, in WO 2009 / 134347 A2 and commercially available under the trade name Captisol®.
- the anthocyanidin used in complexed form according to the invention is delphinidin.
- the chemical structure corresponds to the formula given above with the following substitution pattern
- the invention further provides an aqueous solution of the composition according to the invention for use as a medicament for the treatment of malignant melanoma.
- the preparation of the complex of the invention as well as a corresponding aqueous solution comprises the following Schrit ⁇ te: a) preparing an aqueous solution of the .beta.-cyclodextrin Sufoalkylether,
- an aqueous solution is preferred
- Herge ⁇ represents containing 5 to 10 wt .-% of the cyclodextrin used. It is particularly preferred in the context of OF INVENTION ⁇ dung if the pH of the aqueous solution during or after, but preferably before the addition of delphinidin to a pH value of 7 or less, preferably 6 or less, more preferably 5 or less , more preferably 4 to 5, is set. It has been shown that at this pH, a higher concentration of the complex can be adjusted in aqueous solution.
- the concentration of delphinidin calculated as chloride preferably at least 0.5 mg / ml, more preference ⁇ example at least 1.0 mg / ml, more preferably at least 1.5 mg / ml, more preferably 2.0 mg / ml.
- the particularly be ⁇ ferred concentration range of at least 2.0 mg / ml can be set in particular in an aqueous solution having a pH between 4 and 5 a preferred embodiment.
- the mixing of the constituents of the aqueous solution can be effected by stirring, preferred periods for mixing are 2 to 20 h. Before ⁇ given to work in the dark, to avoid a Lichtinduz (7).
- Another object of the invention is a solid for use as a medicament in the treatment of malignant melanoma, which is obtainable according to the invention by removing the solvent from an inventive aqueous solution described above ⁇ .
- the removal can preferably be carried out by freeze-drying (lyophilization).
- Both the aqueous drug solution of the invention and the drug solids have high storage stability ⁇ formality.
- Figure 1 shows histograms of delphinidin-SBE- ⁇ -CD treated (b) and untreated (a) cells of the human melanoma cell line A-375. shows the dose-dependent apoptosis induction determined by means of the sub-Gl peak method
- FIG. 5 shows cell number and cell proliferation (real-time cell monitoring) of delphinidin-SBE- ⁇ -CD-treated A-375 cells of high cell confluency in FIG Comparison to untreated cells with the XCELLLigence system.
- FIG. 6 shows the cell count and cell proliferation (real-time cell monitoring) of delphinidin-SBE- ⁇ -CD-treated A-375 cells of low cell confluency compared to untreated cells with the XCELLLigence system.
- FIG. 7 shows cell counts and cell proliferation (real-time cell monitoring) of delphinidin Cl-treated A-375 cells of high cell confluence compared to untreated cells with the system XCELLLigence.
- FIG. 8 shows cell counts and cell proliferation (real-time cell monitoring) of delphinidin Cl-treated A-375 cells with low cell confluence compared to untreated cells with the XCELLLigence system.
- FIG. 9 shows the cell count and cell proliferation (real-time cell monitoring) of SBE- ⁇ -CD-treated A-375 cells of high cell confluence compared to untreated cells with the XCELLLigence system.
- FIG. 10 shows the cell count and cell proliferation (real-time cell monitoring) of SBE- ⁇ -CD-treated A-375 cells of low cell confluence compared to untreated cells with the XCELLLigence system.
- FIG IIa shows the erstoff by means of sub-Gl-peak method ⁇ te apoptosis induction by delphinidin SBE-ß-CD and without TRAIL compared to the Kontrol ⁇ len (untreated cells or only with TRAIL-treated cells) at low cell confluency.
- FIG IIb shows the erstoff by means of sub-Gl-peak method ⁇ te apoptosis induction by delphinidin SBE-ß-CD with or without TRAIL compared to the Kontrol ⁇ len (untreated cells or only with TRAIL, cells treated with SBE-ß -CD treated cell h ⁇ len) at high cell confluency.
- FIG. 12a shows the cell viability effect of delphinidin-SBE- ⁇ -CD with TRAIL as determined by WST-1 assays in comparison with delphinidin-SBE- ⁇ -CD alone and the controls (untreated cells or TRAIL-treated cells only) nied ⁇ engined cell confluence.
- FIG. 12b shows the cell viability effect of delphinidin-SBE- ⁇ -CD with TRAIL as determined by WST-1 assays in comparison with the delphinidin-SBE- ⁇ -CD alone and the controls (untreated cells or TRAIL-treated cells only) SBE-ß-CD-treated cells) at high cell ⁇ confluence.
- FIG. 13 shows micrographs of non-commercially ⁇ ten, treated with TRAIL, treated only with delphinidin SBE-ß-CD, and treated with delphinidin SBE-ß-CD and TRAIL A-375 cells
- FIG. 14 shows the cell count and cell proliferation (real-time cell monitoring) of A-375 cells of high cell confluence treated with TRAIL, delphinidin-SBE- ⁇ -CD, TRAIL and delphinidin-SBE- ⁇ -CD in comparison with untreated cells with the XCELLLigence system ,
- Figure 15 shows cell counts and cell proliferation (real-time cell monitoring) of high cell confluency A-375 cells treated with TRAIL, delphinidin Cl, TRAIL and delphinidin Cl compared to untreated cells with the XCELLLigence system.
- FIG. 16 shows cell number and cell proliferation (real-time cell monitoring) of TRAIL (0.02 pg / ml), SBE- ⁇ -CD (30 pg / ml), TRAIL (0.02 pg / ml) and SBE- ⁇ -CD (30 pg / ml) treated high cell confluency A-375 cells compared to untreated cells with the XCELLLigence system.
- Figure 17 shows cell number and cell proliferation (real-time cell monitoring) of TRAIL (0.02 pg / ml), SBE- ⁇ -CD (1000 pg / ml), TRAIL (0.02 pg / ml) and SBE- ⁇ -CD (1000 pg / ml) treated high cell confluency A-375 cells compared to untreated cells with the XCELLLigence system.
- FIG. 18 shows the effect of delphinidinCl
- the column used was a Waters X Bridge TM C18, 35 ⁇ , 150 mm x 4.6 mm.
- the mobile phases were as follows:
- Channel A water 950 ml, methanol 50 ml, formic acid 10 ml
- Channel B water 50 ml, methanol 950 ml, formic acid 10 ml
- UV-Vis detector 530 pm for the assay, 275 pm for the detection of contaminants
- Dilution solution 1 Mixture of 100 ml of methanol and 2.6 ml of M HCl
- a reference solution of delphinidin was prepared by weighing 10 mg delphinidin chloride into a 10 ml flask and dissolving in dilution solution 1. After dissolution, it was diluted approximately 10-fold with dilution solution 2 to make an approximate concentration of 0.1 mg / ml.
- control calibration solution was prepared in the same manner.
- the calibration solutions were analyzed immediately by HPLC, since delphinidin chloride is unstable in solution.
- the suspensions were stirred for 20 h at 30 ° C in the dark.
- the mixture was then filtered through a Membranfil ⁇ ter of 0.22 pm pore size.
- Example 2 Influence of the pH
- the influence of pH on the solubility of a delphinidin-SBE- ⁇ -CD in aqueous solution was investigated.
- aqueous solutions of SEB- ⁇ -CD were prepared, but these solutions were adjusted to the acidic pH values indicated in Table 2 using 1 M HCl.
- delphinidin chloride was added according to the instructions of Example 1 and wentge ⁇ works, the only deviation, the stirring time was limited to 2.5 h. The results are reproduced in the following Tabel ⁇ le.
- a complex of the invention is formulated as a solid.
- a complex of the invention is formulated as a solid.
- Example 3.1 It was worked in the same manner as Example 3.1, but ⁇ a significant amount of material was filtered off ⁇ filtration, suggesting that the solubilization was significantly less effective than when using SBE-ß-CD according to Example 3.1 ,
- Example 3.1 is according to the invention, examples 3.2 and 3.3 are comparative examples.
- the last 22 days of the storage described above were carried out in glass vials with a volume of 20 ml. 250 mg each of the previously already stored for 8 days Pro ⁇ ben were charged there, the vials were sealed with a rubber stopper and sealed. Using two hypodermic needles, the headroom of the violas was flushed with pure oxygen. The samples were then stored in Dun ⁇ angles.
- the delphinidine content of the solids (calculated as dolphinidinium chloride and reported in wt%) was determined by the HPLC method described above. The results can be found in the following table.
- the column used was a Waters X Bridge TM C18, 35 ⁇ , 150 mm x 4.6 mm.
- the mobile phases were as follows:
- Channel A water 770 ml, methanol 230 ml, formic acid 10 ml
- Channel B water 50 ml, methanol 950 ml, formic acid 10 ml
- UV-Vis detector 530 pm for the assay, 275 pm for the detection of contaminants
- Dilution solution 1 Mixture of 100 ml of methanol and 2.6 ml of 1 M HCl
- Calibration solution A reference solution of delphinidin was prepared by weighing 10 mg delphinidin chloride into a 10 ml flask and dissolving in dilution solution 1. After dissolution, it was diluted approximately 10-fold with dilution solution 2 to make an approximate concentration of 0.1 mg / ml.
- control calibration solution was prepared in the same manner.
- the calibration solutions were analyzed immediately by HPLC, since delphinidin chloride is unstable in solution.
- the delphinidine content was determined.
- the following table indicates the determined content as a percentage of the above-mentioned starting concentration.
- delphinidin-SBE-.beta.-CD delphinidin-SBE-.beta.-cyclodextrin
- delphinidin-SBE-.beta.-CD delphinidin-SBE-.beta.-CD
- delphinidin-SBE-.beta.-CD delphinidin-SBE-.beta.-CD
- delphinidin-SBE-.beta.-CD delphinidin-SBE-.beta.-CD
- the cell line was cultured at 37 ° C, 5% C0 2 in DMEM (Dulbecco's Modified Eagle's medium from Gibco, Düsseldorf, Germany) supplemented with 10% FCS (Fetal Bovine Serum) and antibiotics.
- DMEM Dulbecco's Modified Eagle's medium from Gibco, Düsseldorf, Germany
- FCS Fetal Bovine Serum
- the method is based on the lower DNA content of apoptotic cells compared to vital cells.
- a feature of apoptosis is the cleavage of the DNA by endonucleases into short DNA fragments, which increases the content of low molecular weight DNA in apoptotic cells compared to vital cells and reduces the proportion of high molecular weight DNA.
- Upon induced lysis (permeabilization) of the cell membrane the low molecular weight DNA fragments escape from the apoptotic cells.
- PI diumiodid with propionaldehyde
- Propidium iodide has been incorporated into the DNA and fluoresces in two narrow peaks, the Gl phase (left peak) with single and the cells in the G2 phase (right peak) with double DNA content.
- a sub-Gi peak upstream of the left peak is nearly absent.
- Histogram b shows a broad subGi peak. This represents the proportion of apoptotic cell nuclei after 24 hours of delphinidin-SBE- ⁇ -CD treatment.
- the cells were incubated for 24 hours with 10-3000 pg delphinidin-SBE- ⁇ -CD per ml cell suspension and 15-120 ⁇ purified delphinidin chloride (hereinafter delphinidin Cl) per incubated with the complex partner SBE-ß-CD and DMSO treated cells, as well as untreated cells as controls.
- delphinidin Cl delphinidin chloride
- the cells were then harvested by trypsinization, washed with ice-cold phosphate buffered saline (PBS) and stained for 1 hour with the staining buffer containing 0.1% sodium citrate, 0.1% Triton X-100 and PI (40pg / ml; Sigma-Aldrich, Taufkirchen , Germany) before subsequently measuring and evaluating the DNA content of the cell nuclei with the flow cytometer (FACSCalibur and CellQuest Software, Becton Dickinson, Heidelberg). From each of the three measured values, the mean value was calculated, with the measured values of the control cells serving as a reference for the t-test.
- PBS ice-cold phosphate buffered saline
- the dolphin-SBE- ⁇ -CD complex shows a markedly strong induction of apoptosis (see Figure 2a at 1000 pg / ml and Figure 2b at 3000 pg / ml);
- the WST-1 Assay serves to detect an intact respiratory chain in cells, viable cells with an intact mitochondrial succinate-tetrazolium dehydrogenase system an enzymatic reaction of the weakly red colored
- Tetrazolium salt WST-1 (4- [3- (4-iodophenyl) -2- (4-nitrophenyl) -2H-5-tetrazolio] -1,3-benzene disulfonate) into the dark red formazan. This color change can be photometrically measured and evaluated in a spectrophotometer.
- Example 7 For the implementation of the WST-I assay as described in Example 7 ⁇ WUR which the cells of Example 6 for 24 hours with 10-3000 pg delphinidin SBE-ß-CD per ml of cell suspension or 15-120 ⁇ purified delphinidin Cl per ml of cell suspension incubated with the complex partners SBE-ß-CD and DMSO ⁇ be acted cells, and untreated cells served as Kontrol ⁇ len.
- the WST-I assay such as in Plötz et al.
- Example 6 Analogously to Example 6, the experiment was also carried out in Example 7 so ⁇ probably at low (low cell concurrence), as well as higher cell density (higher cell concurrency), the results in Figures 3a (low cell confluence) and 3b (higher cell concurrence) are presented graphically processed ,
- Cell vitality is effected by the delphinidin-SBE- ⁇ -CD complex (see Figure 3a at 1000 pg / ml and 2000 pg / ml and Figure 3b at 3000 pg / ml);
- Example 8 The effect of loss of vital cells beyond the induction of apoptosis (Example 6) is also reflected in the morphology of the cells when examined microscopically for 24 hours, which precipitate at high concentrations of delphinidin Cl or delphinidin-SBE- ⁇ -CD and / or peel off, as can be seen from the microscopic images in Figure 4.
- Example 8 The effect of loss of vital cells beyond the induction of apoptosis (Example 6) is also reflected in the morphology of the cells when examined microscopically for 24 hours, which precipitate at high concentrations of delphinidin Cl or delphinidin-SBE- ⁇ -CD and / or peel off, as can be seen from the microscopic images in Figure 4.
- Example 8 Example 8
- RTCA Continuous real-time cell analysis
- the cell count and cell proliferation with the same drug study analogous to Examples 6 and 7 was recorded in real time by means of the system xCELLLigence (Roche Diagnostics, Mannheim, Germany).
- the xCELLigence Sys ⁇ tem is a microelectronic biosensor for zellba ⁇ catalyzed, label-free analysis, provides the dynamic cell data in real time.
- the culture plates of xCELLigence Sys tems ⁇ are equipped each well with microelectrodes on the ground, in order to measure changes in electrical impedance. Quantitative impedance changes correlate with the number and strength of cell contacts with the underlying electrodes.
- Delphinidin-SBE-ß-CD shows in accordance with the high rates of apoptosis (Example 6) and the loss of Zellvita ⁇ lity (Example 7) a complete collapse of the
- Cell proliferation in particular strong antiproliferative effects from a concentration of 1000 pg / ml in Figure 5 and a complete blockade of cell proliferation from 500 pg / ml in Figure 6.
- the effect of the delphinidin-SBE- ⁇ -CD complex in combination with the pro-apoptotic death ligand TRAIL, against which the cell line A-375 possesses only moderate apoptosis sensitivity was investigated by means of the sub-Gi peak method .
- the cells were incubated for 24 hours with 30-1000 pg of delphinidin-SBE- ⁇ -CD per ml of cell suspension with and without 0.02 pg of TRAIL per ml of cell suspension the complex partners SBE-ß-CD-treated cells served as controls carried out as ⁇ and untreated cells.
- the experiments were carried out in parallel both at low (low cell confluence), and higher cell density (higher cell concurrency), the result ⁇ se in the figures IIa (low cell concurrence) and IIb (higher cell concurrency) are shown graphically processed.
- Example 10 the cell viability of the test cell line was unersucht analogously to Example 7, with the Un ⁇ ter Kunststoff that the cells of the combination of Delphinidin- SBE-ß-CD complexes and TRAIL were suspended.
- the cells were incubated for 24 hours with 30-1000 pg of delphinidin-SBE- ⁇ -CD per ml of cell suspension with and without 0.02 pg / ml TRAIL per ml of cell suspension, the complex partner SBE-ß CD-treated cells, as well as untreated cells served as controls.
- Example 9 Analogously to Example 9, the experiment was so ⁇ well performed at low (low cell confluency) as well as higher cell density (higher cell confluency) in Example 10, the results displayed graphically in Figures 12a (low cell confluency) and 12b (higher cell confluency) Darge ⁇ are.
- RTCA Continuous real-time cell analysis
- Delphinidin-SBE-ß-CD is in combination with TRAIL in accordance with the opposite delphinidin SBE-ß-CD alone increased apoptosis rates (Example 9) as well as the greater loss of cell viability (Example 10) a sy ⁇ nerg Vietnamese and greater blockage of cell proliferation (see Figure 14).
- a stronger block of cell proliferation can also be found in the combination of delphinidin Cl with TRAIL against TRAIL alone and delphinidin alone (see Figure 15).
- the cell structure of the tissue was loosened by a one-hour incubation with trypsin-EDTA solution.
- stop medium was added to the skin.
- the piece of skin was swung twice in PBS, so that a fibroblast suspension was obtained.
- the fibroblasts obtained were centrifuged off at 4 ° C. for 5 min at 1000 revolutions and used after quantification.
- the intracellular ATP content decreases strongly by ATPase-mediated degradation.
- the liberated ATP content can thus be used as a measure of the number of vital cells.
- the released ATP is coupled to a reaction catalyzed by the enzyme luciferase, in which, in the presence of Mg 2+, the ATP-dependent oxidation of the substrate luciferin to oxyluciferin, carbon dioxide, AMP, inorganic phosphate with release of light takes place.
- the amount of light released correlated with the ATP content is measured and allows an indirect quantifiable inference to the vitality of the examined cells.
- RLU Relative Light Units
- Kits and devices for ATP determination are known in the art, for example, the ATP test kit from. Biothema AB, Haning, Sweden, and the portable luminometer Lumino® Fa. STRATEC Biomedical Systems AG, Birkenfeld, Germany.
- To determine the intracellular ATP usually 50 .mu. ⁇ of the sample to be examined in a Pippettiert glass tube, to 50 ⁇ of Extractant- reagent B / S for cell lysis and 400 ⁇ of the ATP-HS reagent added and added after subsequent mixing the striglos ⁇ sene tube in the luminometer for automatic measurement and evaluation of the light emission.
- the human fibroblast or endothelial cells Prior to the drug treatment and application of the ATP luminescence assay in the present case, the human fibroblast or endothelial cells were seeded with Dulbecco's Modified Eagle's Medium (DMEM medium) on 96-well microtiter plates (5000 cells / well) and for Cultivated for 3 days. After this Anheftungs- and growth phase of the cells in the incubator cabinet ⁇ substance treatment took place at time intervals from day 1-day 4 (endothelial cells) and 4h, 24h, 48h and 96h
- DMEM medium Dulbecco's Modified Eagle's Medium
- the active substance delphinidinCl or delphinidin-SBE- ⁇ -CD complex dissolved in 3 wells in ⁇ DMEM medium was added in different concentrations (0.1 ⁇ , 3.2 ⁇ , 100 ⁇ ).
- test results of the ATP luminescence assays are shown in Figures 18 (endothelial cells) and 19 (fibroblasts) graphically processed and can be as follows
- Figures 18 and 19 fibroblasts graphically processed and can be as follows
- DelphinidinCl and delphinidin-SBE-ß-CD show no significant cytotoxic effects on human at the investigated drug concentration (0.1-100 ⁇ )
- Fibroblasts and endothelial cells This opens up a completely new and particularly geeig ⁇ designated agent group opens for cancer treatment, the tested drugs DelphinidinCl and delphinidin-SBE-.beta.-CD have but one hand the desired antiproliferative effects on cancer cells (melanoma cells) to (examples 6-11), but avoid the other hand, the expected therapeutic agent at concentrations ⁇ usually undesirable cytotoxic (side) effects on non-cancer cells based on the present example 12.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
- Nanotechnology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Inorganic Chemistry (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Medical Informatics (AREA)
- Crystallography & Structural Chemistry (AREA)
Abstract
The invention relates to a composition comprising a complex of delphinidin and a sulfoalkyl ether-β-cyclodextrin for use in the treatment of malignant melanoma.
Description
DELPHINIDIN ZUR BEKÄMPFUNG VON MELANOMZELLEN DELPHINIDINE FOR COMBATING MELANOMA CELLS
Die Erfindung betrifft Zusammensetzungen umfassend The invention relates to compositions comprising
- einen Komplex aus Delphinidin und einem Sulfoalkylether- ß-Cyclodextrin und/oder a complex of delphinidin and a sulfoalkylether-β-cyclodextrin and / or
- Delphinidin oder dessen Salze - Delphinidin or its salts
zur Verwendung bei der Behandlung von malignem Melanom. for use in the treatment of malignant melanoma.
Bei dem malignen Melanom, auch unter der Bezeichnung „schwarzer Hautkrebs" bekannt, handelt es sich um eine bös¬ artige Entartung der Pigment zellen, den sogenannten Melanozyten. Der Krebs neigt bereits in einem frühen Stadium zum Streuen von Metastasen über Blut- und Lymphbahnen. Das maligne Melanom ist heilbar, jedenfalls dann, wenn es früh diagnostiziert und behandelt wird. In the malignant melanoma, also known as "black skin cancer" known, it is a wicked ¬-like transformation of the pigment cells, called melanocytes. The cancer already inclined at an early stage for spreading metastases through blood and lymph vessels. Malignant melanoma is curable, at least if it is diagnosed and treated early.
Die wichtigste Behandlungsmethode ist die chirurgische Ent¬ fernung des Tumors neben der Strahlentherapie, obgleich auch die Monotherapie mit Interferonen bei der Behandlung des malignen Melanoms Anwendung findet. Ebenfalls bekannt ist die Vakzintherapie, d.h. die aktive Immuntherapie mit¬ tels Tumorimpfungen unter Verwendung von Tumorvakzinen um die körpereigene Abwehr zur gezielten Bekämpfung von Krebszellen anzuregen. Den Abwehrzellen werden dabei über die verabreichten Vakzine Merkmale des Tumors, (beispielsweise Eiweißmoleküle, die von den Tumorzellen produziert werden oder Zellbruchstücke der Tumorzellen) so präsentiert, dass die Abwehrzellen diese Merkmale als fremd erkennen und kör¬ pereigenen Zellen, die diese Merkmale tragen, angreifen.
Neben der Vakzintherapie ist die Chemotherapie, d.h. die medikamentöse Behandlung mit chemischen Substanzen, die Tumorzellen schädigen oder hemmen (Zytostatika), ebenfalls von praktischer Relevanz. The main method of treatment is surgical Ent ¬ fernung the tumor alongside radiotherapy, although monotherapy with interferons in the treatment of malignant melanoma applies. Also known vaccine therapy, ie the active immunotherapy with ¬ means tumor vaccines using tumor vaccines to stimulate the body's defense against targeted control of cancer cells. The immune cells are characterized by the administered vaccine features of the tumor, (for example, protein molecules that are produced by the tumor cells or cell fragments of tumor cells) presented so that the immune cells recognize these features as foreign and kör ¬ pereigene cells that carry these features attack , In addition to vaccine therapy, chemotherapy, ie drug treatment with chemical substances that damage or inhibit tumor cells (cytostatics), is also of practical relevance.
Aufgabe der vorliegenden Erfindung ist es, ein wirksames Mittel als Alternative oder Ergänzung zu aus dem Stand der Technik bekannten Behandlungsmöglichkeiten des malignem Melanoms zur Verfügung zu stellen. The object of the present invention is to provide an effective means as an alternative or supplement to treatment options of the malignant melanoma known from the prior art.
Gelöst wird diese Aufgabe durch die in den unabhängigen An¬ sprüchen genannten Zusammensetzungen und Verwendungen, wobei vorteilhafte Ausführungsformen der Erfindung in den Unteransprüchen offenbart sind. Dass diese Aufgabe in der Tat gelöst wird, belegen die in-vitro Versuchsergebnisse zu der Wirkung der erfindungsgemäßen Zusammensetzungen auf Melanomzellen und Nicht-Krebszellen und die Zellvitalität in den Beispielen 6- 12. This object is solved by the independent ¬ in the claims referred to compositions and uses wherein advantageous embodiments of the invention are disclosed in the subclaims. That this problem is indeed solved, the in vitro test results to the effect of the compositions of the invention on melanoma cells and non-cancerous cells and the cell vitality in Examples 6-12.
Zunächst seien einige im Rahmen der Erfindung verwendete Begriffe erläutert. First, some terms used in the invention are explained.
Die erfindungsgemäße Zusammensetzung wird zur Behandlung eines Objekts oder Individuums eingesetzt, das am malignem Melanom leidet. Der Begriff „Objekt" umfasst lebende Tiere und Menschen. Der Zweck dieser Behandlung ist die zumindest teilweise Abtötung oder Neutralisation der Tumorzellen. „Neutralisation" und „Abtötung" bedeuten im Sinne der vorliegenden Erfindung die zumindest teilweise Zerstörung oder Auflösung oder Inaktivierung oder Vermehrung der Tumorzellen .
Die vorliegende Erfindung bezieht sich auch auf Verfahren zur Behandlung eines an malignem Melanom leidenden Objekts, wobei dem Objekt eine therapeutisch wirksame Menge der er¬ findungsgemäßen Zusammensetzung verabreicht wird. Von besonderen Interesse ist es dabei, einerseits die Melanomzel- len mit den Wirkstoff zu bekämpfen, andererseits aber auch weitest möglich die Vitalität und Proliferation der Nicht¬ Krebszellen unbeeinträchtigt zu lassen. The composition of the invention is used to treat an object or individual suffering from malignant melanoma. The term "object" includes live animals and humans The purpose of this treatment is the at least partial killing or neutralization of the tumor cells. "Neutralization" and "killing" in the sense of the present invention mean the at least partial destruction or dissolution or inactivation or multiplication of the tumor cells , The present invention also relates to methods of treating a patient suffering from malignant melanoma object, wherein the object comprises a therapeutically effective amount of he ¬ inventive composition is administered. Of particular interest here is to on the one hand Melanomzel- len fight with the drug, but then possible to have too much as the vitality and proliferation of non ¬ cancer cells unaffected.
Der Begriff „Behandlung" bedeutet im Sinne der vorliegenden Erfindung das ganze oder teilweise Erreichen der nachfol¬ gend genannten Ergebnisse: Ganze oder teilweise Reduktion des Krankheitsbildes; Verbesserung mindestens eines der klinischen Symptome oder mit der Krankheit assoziierten Indikatoren; Verzögerung, Unterdrückung oder Schutz vor dem Fortschreiten der Krankheit; oder ganze oder teilweise Verzögerung, Unterdrückung oder Schutz vor dem Ausbrechen oder Entstehung der Krankheit. Das zu behandelnde Objekt ist ein Mensch oder Tier, vorzugsweise ein Säugetier. Die veterinärmedizinische Behandlung umfasst neben der Behandlung von Nutz- oder Wildtieren (z.B. Schafe, Katzen, Pferde, Kühe, Schweine) auch Labortiere (z.B. Ratten, Mäuse, Meerschwei¬ ne, Affen) . In the sense of the present invention, the term "treatment" means the whole or part of reaching the nachfol ¬ quietly mentioned results: the whole or partial reduction of the disease, improvement of at least one of the clinical symptoms or associated with the disease indicators, delay, suppression or protection against Disease progression, or partial or total delay, suppression or protection against the onset or onset of the disease The object to be treated is a human or animal, preferably a mammal Veterinary treatment includes treatment of beneficial or wild animals (eg sheep , cats, horses, cows, pigs), laboratory animals (eg rats, mice, guinea pig ¬ ne, monkeys).
Der Begriff „Zusammensetzung umfassend einen Komplex aus Delphinidin und einem Sulfoalkylether-ß-Cyclodextrin und/oder Delphinidin oder dessen Salze" schließt die Zusammensetzung als Monopräparat , d.h. ohne weitere therapeu¬ tisch aktive Komponenten ein. Alternativ kann die Zusammensetzung mindestens eine weitere therapeutisch aktive Sub¬ stanz umfassen. Die erfindungsgemäße Zusammensetzung kann allein oder in Kombination mit mindestens einer anderen therapeutischen Substanz zur Verringerung eines oder mehre-
rer Symptome des malignen Melanoms verabreicht werden. Die Verabreichung der erfindungsgemäßen Zusammensetzung kann gleichzeitig mit der anderen therapeutischen Substanz, welche Bestandteil derselben Zusammensetzung sein kann oder in einer anderen Zusammensetzung bereitgestellt wird, verabreicht werden. Alternativ kann die erfindungsgemäße Zusammensetzung vor oder nach der Verabreichung der anderen therapeutischen Substanz verabreicht werden. Die erfindungsge¬ mäße Zusammensetzung kann über den gleichen oder einen anderen Verabreichungsweg wir die andere therapeutische Sub¬ stanz verabreicht werden. The term "composition comprising a complex of delphinidin and a sulfoalkyl ether-.beta.-cyclodextrin and / or delphinidin or its salts" includes the composition as a single preparation, ie without further therapeutically ¬ schematically active components. Alternatively, the composition may be at least one other therapeutically active Sub ¬ substance include. The composition of the invention may be used alone or in combination with at least one other therapeutic agent for the reduction of one or several symptoms of malignant melanoma. Administration of the composition of the invention may be administered concomitantly with the other therapeutic substance, which may be part of the same composition or provided in a different composition. Alternatively, the composition of the invention can be administered before or after administration of the other therapeutic substance. The erfindungsge ¬ Permitted composition may be administered via the same or a different route of administration, we the other therapeutic Sub ¬ substance.
Die weitere therapeutisch wirksame Substanz ist vorzugswei¬ se ein Tumornekrosefaktor-verwandter Apoptose-induz ierter Ligand (Tumor Necrosis Factor Related Apoptosis I_nducing Ligand - TRAIL) . Gemäß einer weiteren bevorzugten Ausführungsform der Erfindung ist die weitere therapeutisch wirksame Substanz ausgewählt aus der Gruppe bestehend aus Zy¬ tostatika, Interferone, vorzugsweise Alpha- und/oder Beta- Interferone, weiter vorzugsweise Interferon alpha 2-a und alpha 2-b, und Tumorvakzine. Letztere können auch in Kombi¬ nation mit TRAIL kombiniert und angewendet werden. The other therapeutically active substance is vorzugswei ¬ se a tumor necrosis factor-related apoptosis-induz FOURTH ligand (Tumor Necrosis Factor Related Apoptosis I_nducing Ligand - TRAIL). According to another preferred embodiment of the invention the further therapeutically effective substance is selected from the group consisting of Zy ¬ tostatika, interferons, preferably alpha and / or beta interferons, more preferably interferon alpha 2-a and alpha 2-b, and tumor vaccines , The latter can also be combined in combi ¬ nation with TRAIL and applied.
Insbesondere wenn sich bereits Metastasen in anderen Organen gebildet haben, können eine Kombination von Chemotherapie und Immuntherapie, sowie eine Strahlenbehandlung ein¬ zelner Metastasen angebracht sein um Rückbildungen zu erreichen . Especially if there is already metastases have formed in other organs, a combination of chemotherapy and immunotherapy, and radiation therapy may be appropriate to achieve a ¬ of individual metastases by regressions.
Bei der Chemotherapie werden üblicherweise Interferone, insbesondere von den weißen Blutkörperchen (Leukozyten) bildetes IFN-alpha, und von den Bindegewebszellen In chemotherapy, interferons, in particular of the white blood cells (leucocytes), IFN-alpha, and connective tissue cells are commonly used
(Fibroblasten) gebildetes IFN-beta, verwendet, die das
Wachstum von gesunden als auch bösartigen Zellen hemmen und das Immunsystem anregen. In der Krebstherapie wird übli¬ cherweise auf reines, gentechnologisch gewonnenes Interfe¬ ron zurückgegriffen, beispielsweise auf das in Deutschland zur Krebsbehandlung zugelassene Interferon alpha 2-a (Rofe- ron®) und Interferon alpha 2-b (IntronA®) . (Fibroblasts) formed IFN-beta, using the Inhibit growth of healthy as well as malignant cells and stimulate the immune system. In cancer therapy übli ¬ cherweise use is made of pure, genetically derived Interfe ¬ ron, for example, on the approved in Germany for cancer treatment interferon alpha 2-a (Rofe- ron®) and interferon alpha 2-b (IntronA®).
Bei der Immuntherapie kommt das ursprüngliche Konzept, bei dem körpereigene Tumorzellen durch Bestrahlung teilungsunfähig gemacht und um den Reiz für das Immunsystem zu erhöhen, vorzugsweise mit einem Virus vermischt, in die Haut gespritzt wird um Abwehrzellen anzulocken und gezielt gegen diese Tumorzellen zu aktivieren, immer seltener zur Anwendung. Statt der Verwendung ganzer Tumorzellen oder Zellbruchstücke wird nunmehr regelmäßig auf von den Tumorzellen produzierte Eiweißmoleküle zurückgegriffen, mit denen aus Blutvorläuferzellen gezüchtete Zellen in-vitro beladen werden und so bestückt dem Patienten zurückgegeben werden um sie den Abwehrzellen als etwas zu präsentieren, was bekämpft werden muss. Alternativ kommt man zu gleichem Ergebnis, wenn in die Tumorzellen das Gen eines Lock- bzw. Aktivierungsstoffes für Abwehrzellen eingeschleust wird. In immunotherapy, the original concept, in which the body's own tumor cells are rendered unable to divide by radiation and to increase the stimulus for the immune system, preferably mixed with a virus is injected into the skin to attract defense cells and targeted to activate these tumor cells, increasingly rare for use. Instead of using whole tumor cells or cell fragments, protein molecules produced by the tumor cells are now regularly used, with which cells grown from blood precursor cells are loaded in vitro and thus returned to the patient to present them to the defense cells as something to be controlled. Alternatively, the same result is obtained when the gene of a blocking or activating substance for defense cells is introduced into the tumor cells.
Von praktischer Relevanz ist ebenfalls die eingangs genannte und gegen Krebszellen gerichtete Chemotherapie bei der verschiedene Medikamente verwendet werden, die das Tumor¬ wachstum auf unterschiedliche Weise hemmen können, wobei diese Chemotherapeutika üblicherweise als „Zytostatika" be¬ zeichnet werden. Zytostatika werden synthetisch hergestellt oder sind Abkömmlinge von in der Natur vorkommenden Zellgiften, die den programmierten Zelltod (Apoptose) der Tumorzellen auslösen. Beispiele für chemotherapeutische Mit¬ tel, welche im Rahmen der vorliegenden Erfindung Verwendung
finden können, schließen Bortezomib (Velcade®, Millennium) , Melphatan, Prednison, Vincristin, Carmustin, Cyclophospha- mid, Dexamethason, Thalidomid, Doxorubicin, Cisplatin, Eto- posid und Cytarabin ein. Of practical relevance is also mentioned at the outset and directed against cancer cells chemotherapy in the different medications are used which can inhibit tumor ¬ growth in different ways, these chemotherapeutic agents are be ¬ features commonly called "cytostatic agents". Cytostatics produced synthetically or Derivatives of naturally occurring cytotoxins which trigger the programmed cell death (apoptosis) of the tumor cells Examples of chemotherapeutic agents which are used in the context of the present invention include bortezomib (Velcade®, Millennium), melphatan, prednisone, vincristine, carmustine, cyclophosphamide, dexamethasone, thalidomide, doxorubicin, cisplatin, estrogen and cytarabine.
Neben der erfindungsgemäßen medikamentösen Verabreichung kann ergänzend eine Strahlentherapie zur Anwendung kommen. Abgesehen von der Verwendung radioaktiver Medikamente, so genannten „Radiopharmaka" , wird die Strahlentherapie vor¬ zugsweise örtlich begrenzt eingesetzt. Das heißt, die vor¬ zugsweise elektromagnetische Strahlung und auch Teilchen¬ strahlen wirken in diesem Fall örtlich begrenzt in dem durchstrahlten Bereich und schädigen die Tumorzellen, insbesondere die DNS im Zellkern durch Ionisation und Entste¬ hung freier Radikale und Brüchen in der DNS der Tumorzellen. Um das den Tumor umgebende gesunde Gewebe zu schützen können Blenden verwendet werden. In addition to the drug administration according to the invention, radiotherapy can additionally be used. Apart from the use of radioactive drugs, so-called "Radiopharmaceuticals", the radiotherapy is used preferably localized before ¬. That is, rays which preferably before ¬ electromagnetic radiation and particles ¬ in this case act locally in the irradiated area and damage the tumor cells, in particular the DNA in the nucleus by ionization and Entste ¬ hung free radicals and breaks in the DNA of tumor cells. can be used to protect the tumor surrounding healthy tissue aperture.
Die Verabreichung des erfindungsgemäßen Wirkstoffs im Wege einer Monotherapie oder in Kombination mit TRAIL oder anderen bzw. weiteren therapeutischen Wirkstoffen, beispielsweise ausgewählt aus der Gruppe bestehend aus Zytostatika, Interferone und Tumorvakzine, kann auch als so genannte re¬ gionale Therapie erfolgen, beispielsweise durch gezielte Einspritzung (z.B. mittels Katheter) in Körperregionen, Körperhöhlen oder in die Blutgefäße der Tumorregion bzw. des Organs, in dem der Tumor sitzt. Eine Durchströmung des betroffenen Bereichs oder des Organs ist auch im Weg einer regionalen Perfusion möglich, bei der das Medikament den Bereich (z.B. Arm oder Bein) oder das Organ unter Abschluss vom übrigen Kreislauf durchströmt und direkt wieder abge¬ leitet wird ohne in den übrigen Körper zu gelangen.
Die erfindungsgemäße Zusammensetzung wird vorzugsweise als pharmazeutische Zusammensetzung bereitgestellt und verab¬ reicht. Der Begriff „pharmazeutische Zusammensetzung" um- fasst ein oder mehrere Wirkstoffe und ein oder mehrere in- nerte Inhaltstoffe, die als Träger für den Wirkstoff bzw. die Wirkstoffe fungieren. Die pharmazeutischen Zusammenset¬ zungen erlauben es, den erfindungsgemäßen Komplex oder die erfindungsgemäße Zusammensetzung oral, rektal, parenteral, einschließlich intraperitoneal, perkutan, subkutan, intramuskulär, intravenös, ophthalmisch, pulmonal oder nasal zu verabreichen. Eine parenterale Verabreichungsform kann beispielsweise eine Tablette, Kapsel, Lösung, Suspension oder Dispersion sein. Eine ophthalmische, pulmonale oder nasale Verabreichungsform kann beispielsweise ein Aerosol, Lösung, Creme, Paste, Lotion, Gel, Salbe, Suspension oder Dispersi¬ on sein. Entsprechende Techniken für die Formulierung und Verabreichung sind aus dem Stand der Technik bekannt, siehe beispielsweise „Remington's Pharmaceutical Sciences" (Mack Publishing Co, Easton Pa.) . Zum Beispiel können die erfindungsgemäßen Zusammensetzungen einem Subjekt intravenös mittels eines pharmazeutisch akzeptablen Trägers (z.B. physiologische Salzlösung) verabreicht werden. Für die Injektion bietet sich eine Formulierung in wässriger Lösung, vorzugsweise in physiologisch akzeptablen Puffern an (z.B. Hanks-Lösung, Ringer-Lösung oder physiologisch gepufferte Salzlösung) . Für die parenterale Verabreichung, einschließlich der intravenösen, subkutanen, intramuskulären und intraperitonealen Verabreichung, kommt ebenfalls eine wäss- rige oder ölige Lösung oder eine FestStoffformulierung in Betracht. Der Anteil des aktiven Wirkstoffs in der pharma¬ zeutischen Zusammensetzung kann variieren und liegt üblicherweise zwischen 2 und 60 Gew.-% der Verabreichungseinheit. Der Wirkstoffanteil ist entsprechend so ausgewählt,
dass eine wirksame Dosis erreicht wird. Gemäß einer bevor¬ zugten Ausführungsform der Erfindung werden das Delphinidin oder dessen Salze und/oder der Komplex aus Delphinidin und dem Sulfoalkylether-ß-Cyclodextrin in einer galenischen Zubereitung zur kontrollierten und/oder zeitverzögerten Freisetzung des Delphinidins eingesetzt. The administration of the active compound according to the invention by means of monotherapy or in combination with TRAIL or other or further therapeutic active ingredients, for example selected from the group consisting of cytostatics, interferons and tumor vaccines, can also be carried out as a so-called re ¬ gionale therapy, for example by targeted injection (For example by means of catheter) in body regions, body cavities or in the blood vessels of the tumor region or the organ in which the tumor sits. A flow through the affected area or the organ is also in the way of a regional perfusion possible in which the drug flows through the area (eg arm or leg) or the organ under completion of the rest of the circuit and directly abge ¬ leads is passed into the rest of the body to get. The present composition is preferably provided as a pharmaceutical composition and verab ¬ ranges. The term "pharmaceutical composition" includes fully one or more active ingredients and one or more domestic nerte ingredients which act as carriers for the active ingredient or active ingredients. The pharmaceutical Zusammenset ¬ tongues allow orally administered to the complex of the invention or the composition according to the invention Rectal, parenteral, including intraperitoneal, percutaneous, subcutaneous, intramuscular, intravenous, ophthalmic, pulmonary or nasal administration A parenteral delivery form may be, for example, a tablet, capsule, solution, suspension or dispersion An ophthalmic, pulmonary or nasal dosage form may, for example an aerosol, solution, cream, paste, lotion, gel, ointment, suspension or dispersion .. Appropriate techniques for formulation and administration are known in the art, see, for example, "Remington's Pharmaceutical Sciences" (Mack Publishing Co, Easton Pa.). For example, the compositions of the invention can be administered to a subject intravenously by means of a pharmaceutically acceptable carrier (eg, physiological saline). For the injection, a formulation in aqueous solution, preferably in physiologically acceptable buffers (eg Hanks solution, Ringer's solution or physiologically buffered saline solution), is suitable. For parenteral administration, including intravenous, subcutaneous, intramuscular and intraperitoneal administration, an aqueous or oily solution or solid formulation is also contemplated. The proportion of the active ingredient in the pharmaceutical ¬ ceutical composition may vary and is usually between 2 and 60 wt .-% of the dosage unit. The proportion of active ingredient is selected accordingly, that an effective dose is achieved. According to a prior ¬ ferred embodiment of the invention the delphinidin or its salts and / or complex of delphinidin and the sulfoalkyl ether-beta-cyclodextrin in a pharmaceutical preparation for the controlled and / or delayed release of the delphinidin be used.
„Salz" oder „pharmazeutisch akzeptables Salz" steht für jegliches unter pharmazeutischen Gesichtspunkten akzeptables Salz einer Verbindung vorliegender Erfindung, welches den pharmazeutisch wirksamen Wirkstoff oder dessen aktives Metabolit nach der Verabreichung freigeben kann. Salze der Zusammensetzungen und Komplexe der vorliegenden Erfindung können von anorganischen oder organischen Säuren und Basen abgeleitet sein. "Salt" or "pharmaceutically acceptable salt" means any pharmaceutically acceptable salt of a compound of the present invention which can release the pharmaceutically active agent or its active metabolite after administration. Salts of the compositions and complexes of the present invention may be derived from inorganic or organic acids and bases.
Das Anthocyanidin Delphinidin kann in „Reinform" oder „gereinigt" verwendet werden, was bedeutet, dass ungewünschte Komponenten entfernt wurden. The anthocyanidin delphinidin can be used in "pure" or "purified", meaning that unwanted components have been removed.
„Anthocyanidine" weisen die nachfolgend wiedergegebene Grundstruktur auf. "Anthocyanidins" have the following basic structure.
Die Substituenten in dieser Formel sind ausgewählt aus der Gruppe bestehend aus Wasserstoff, Hydroxygruppe und Metho- xygruppe .
Cyclodextrine, die mit dem Anthocyanidin Delphinidin erfindungsgemäß komplexiert sein können, sind zyklische Oligo¬ saccharide aus CC-1 , 4-glykosidisch verknüpften Glukosemole¬ külen. ß-Cyclodextrin besitzt sieben Glukoseeinheiten. Bei einem Sulfoalkyether-ß-Cyclodextrin sind Hydroxygruppen der Glukoseeinheit in einem Sulfoalkylalkohol verethert . Erfin¬ dungsgemäß ist in der Regel lediglich ein Teil der 21 Hydroxygruppen eines ß-Cyclodextrins verethert. Die Her¬ stellung von Sulfoalkyethercyclodextrinen ist dem Fachmann geläufig und beispielsweise in US 5,134,127 oder WO The substituents in this formula are selected from the group consisting of hydrogen, hydroxy group and methoxy group. Cyclodextrins that can be complexed according to the invention with the anthocyanidin, delphinidin, are cyclic oligo saccharide ¬ from CC-1, 4-glycosidically linked Glukosemole ¬ cules. ß-Cyclodextrin has seven glucose units. In a sulfoalkyl ether-β-cyclodextrin, hydroxy groups of the glucose unit are etherified in a sulfoalkyl alcohol. OF INVENTION ¬ dung according usually only a part of hydroxyl groups of a 21 beta-cyclodextrin is etherified. The Her ¬ position of Sulfoalkyethercyclodextrinen is familiar to the expert and, for example, in US 5,134,127 or WO
2009/134347 A2 beschrieben. 2009/134347 A2.
Sulfoalkyethergruppen werden bei Cyclodextrinen im Stand der Technik zur Erhöhung der Hydrophilie beziehungsweise Wasserlöslichkeit eingesetzt. Sulfoalkylethergruppen tragen in besonderem Maße zur Erhöhung der Stabilität des Komplexes aus Anthocyanidinen und dementsprechend substituiertem ß-Cyclodextrin bei, so dass damit die Lagerstabilität und Formulierbarkeit der besonders oxidat ionsempfindlichen Anthocyanidine wesentlich verbessert wird. Der erfindungs¬ gemäße Komplex kann als lagerstabile wässrige Lösung oder Feststoff formuliert werden, wie unten noch näher gezeigt werden wird. Sulfoalkyl ethers are used in cyclodextrins in the prior art to increase the hydrophilicity or water solubility. Sulfoalkyl ether groups contribute in particular to increase the stability of the complex of anthocyanidins and correspondingly substituted .beta.-cyclodextrin, thus substantially improving the storage stability and formability of the particularly oxidation-sensitive anthocyanidins. The Invent ¬ modern complex can be formulated as a storage-stable aqueous solution or solid, will be shown in more detail below.
Erfindungsgemäß besonders bevorzugt ist die Komplexierung des Wirkstoffs Delphinidin mit einem Sulfoalkylether-ß- Cyclodextrin, vorzugsweise einem Sulfobutylether-ß- Cyclodextrin (SBE-ß-CD) oder einem Sulfoethylether-ß- Cyclodextrin, was überraschenderweise die Löslichkeit und Stabilität des Wirkstoffs erhöht. Ein den Schutzbereich nicht beschränkender Erklärungsversuch hierfür ist, dass die negativ geladenen Sulfobutyleinheiten bzw. Solfoethy- leinheiten mit den positiv geladenen Anthocyanidin Delphi-
nidin elektrostatisch Wechselwirken und unter den Al- kylgruppen die Butylgruppe bzw. Ethylgruppe die optimale Länge besitzen, um sterisch eine entsprechende Wechselwirkung zu ermöglichen. An dieser Stelle sei angemerkt, dass keine allgemein gültige Aussage getroffen werden kann, dass ein beliebiger Wirkstoff, beispielsweise Delphinidin, im Komplex mit einem Sulfoalkylether-ß-Cyclodextrin zu einer Verbesserung der Löslichkeit und Stabilität führt. Bei¬ spielhaft verwiesen sei an dieser Stelle auf Tabelle 1 in Ueda et al . , „Evaluation of a Sulfobutyl Ether ß- Cyclodextrin as a Solubizing/Stabilizing Agent for Several Drugs", Drug Development and Industrial Pharmacy, 24(9), 863-867 (1998), in der die Löslichkeiten verschiedener Wirkstoffe allein, im Komplex mit Sulfobotylether-ß- Cyclodextrin und im Komplex mit ß-Cyclodextrin gegenübergestellt sind. Aus den dort dargestellten Löslichkeit swerten (SBE7-ß-CD vs . ß-CD) erkennt man, dass bei einem Drittel der untersuchten Wirkstoffe im Komplex mit Solfobotylether- ß-Cyclodextrin genau das Gegenteil der Fall ist, d.h. der Komplex aus Wirkstoff und Sulfobotylether-ß-Cyclodextrin eine signifikant geringere Löslichkeit gegenüber dem Kom¬ plex mit ß-Cyclodextrin zur Folge hat. Particularly preferred according to the invention is the complexation of the active substance delphinidin with a sulfoalkyl ether-.beta.-cyclodextrin, preferably a sulfobutyl ether-.beta.-cyclodextrin (SBE-.beta.-CD) or a sulfoethyl ether-.beta.-cyclodextrin, which surprisingly increases the solubility and stability of the active ingredient. An explanatory attempt that does not restrict the scope of protection for this is that the negatively charged sulfobutyl units or solfoethylene units with the positively charged anthocyanidin Delphi nidin interact electrostatically and, among the alkyl groups, the butyl group or ethyl group have the optimum length in order to allow sterically a corresponding interaction. It should be noted that no general statement can be made that any drug, for example delphinidin, in complex with a sulfoalkyl ether-β-cyclodextrin results in an improvement in solubility and stability. In ¬ way of example reference is made at this point to Table 1 in Ueda et al. , "Evaluation of a Sulfobutyl Ether β-Cyclodextrin as a Solubilizing / Stabilizing Agent for Several Drugs", Drug Development and Industrial Pharmacy, 24 (9), 863-867 (1998), in which the solubilities of various drugs alone, in complex with Sulfobotyl ether-β-cyclodextrin and in complex with β-cyclodextrin, and from the solubility values shown there (SBE7-β-CD vs. β-CD) it can be seen that in one third of the active substances investigated in complex with solvobotyl ether β-cyclodextrin. Cyclodextrin exactly the opposite is the case, ie, the complex of active ingredient and sulfobotyl ether-ß-cyclodextrin has a significantly lower solubility compared to Kom ¬ plex with ß-cyclodextrin result.
Bevorzugt beträgt der Substitutionsgrad des Cyclodextrins mit Sulfoalkylethergruppen 3 bis 8, weiter vorzugsweise 4 bis 8, weiter vorzugsweise 5 bis 8, weiter vorzugsweise 6 bis 7. Geeignete Sulfobutylether-ß-Cyclodextrine mit einem mittleren Substitutionsgrad von 6 bis 7 sind beispielsweise in der WO 2009/134347 A2 beschrieben und unter dem Handelsnamen Captisol® kommerziell erhältlich. Ebenfalls verwend¬ bar sind entsprechende Cyclodextrine mit einem Substituti¬ onsgrad von 4 bis 5, beispielsweise 4,2.
Das erfindungsgemäß in komplexierter Form verwendete Antho- cyanidin ist Delphinidin. Die chemische Struktur entspricht der oben wiedergegebenen Formel mit dem folgenden Substitutionsmuster The degree of substitution of the cyclodextrin with sulfoalkyl ether groups is preferably 3 to 8, more preferably 4 to 8, more preferably 5 to 8, further preferably 6 to 7. Suitable sulfobutyl ether-β-cyclodextrins with an average degree of substitution of 6 to 7 are described, for example, in WO 2009 / 134347 A2 and commercially available under the trade name Captisol®. Also verwend ¬ bar corresponding cyclodextrins with a Substituti ¬ onsgrad 4-5, for example, 4.2. The anthocyanidin used in complexed form according to the invention is delphinidin. The chemical structure corresponds to the formula given above with the following substitution pattern
Gegenstand der Erfindung ist ferner eine wässrige Lösung der erfindungsgemäßen Zusammensetzung zur Verwendung als Medikament zur Behandlung von malignem Melanom. The invention further provides an aqueous solution of the composition according to the invention for use as a medicament for the treatment of malignant melanoma.
Die Herstellung des erfindungsgemäßen Komplexes sowie einer entsprechend wässrigen Lösung umfasst die folgenden Schrit¬ te : a) Herstellen einer wässrigen Lösung des Sufoalkylether-ß- Cyclodextrins , The preparation of the complex of the invention as well as a corresponding aqueous solution comprises the following Schrit ¬ te: a) preparing an aqueous solution of the .beta.-cyclodextrin Sufoalkylether,
b) Zugabe des Anthocyanidin Delphinidin und Vermischen zur Herstellung des Komplexes. b) Addition of the anthocyanidin delphinidin and mixing to prepare the complex.
In Schritt a) wird bevorzugt eine wässrige Lösung herge¬ stellt, die 5 bis 10 Gew.-% des verwendeten Cyclodextrins enthält. Besonders bevorzugt ist es im Rahmen der Erfin¬ dung, wenn der pH-Wert der wässrigen Lösung während oder nach, bevorzugt jedoch vor der Zugabe des Delphinidins auf einen pH-Wert von 7 oder weniger, vorzugsweise 6 oder weniger, weiter vorzugsweise 5 oder weniger, weiter vorzugsweise 4 bis 5, eingestellt wird. Es hat sich gezeigt, dass bei diesem pH-Wert sich eine höhere Konzentration des Komplexes in wässriger Lösung einstellen lässt.
Die Konzentration des Delphinidins berechnet als Chlorid, beträgt vorzugsweise wenigstens 0,5 mg/ml, weiter vorzugs¬ weise wenigstens 1,0 mg/ml, weiter vorzugsweise wenigstens 1,5 mg/ml, weiter vorzugsweise 2,0 mg/ml. Der besonders be¬ vorzugte Konzentrationsbereich von wenigstens 2,0 mg/ml lässt sich im Rahmen einer bevorzugten Ausführungsform insbesondere einstellen in einer wässrigen Lösung mit einem pH-Wert zwischen 4 und 5. In step a) an aqueous solution is preferred Herge ¬ represents containing 5 to 10 wt .-% of the cyclodextrin used. It is particularly preferred in the context of OF INVENTION ¬ dung if the pH of the aqueous solution during or after, but preferably before the addition of delphinidin to a pH value of 7 or less, preferably 6 or less, more preferably 5 or less , more preferably 4 to 5, is set. It has been shown that at this pH, a higher concentration of the complex can be adjusted in aqueous solution. The concentration of delphinidin calculated as chloride, preferably at least 0.5 mg / ml, more preference ¬ example at least 1.0 mg / ml, more preferably at least 1.5 mg / ml, more preferably 2.0 mg / ml. The particularly be ¬ ferred concentration range of at least 2.0 mg / ml can be set in particular in an aqueous solution having a pH between 4 and 5 a preferred embodiment.
Im Rahmen der Herstellung kann das Vermischen der Bestandteile der wässrigen Lösung durch Rühren geschehen, bevorzugte Zeiträume für das Vermischen sind 2 bis 20 h. Bevor¬ zugt wird im Dunkeln gearbeitet, um eine lichtinduz ierte Oxidation zu vermeiden. In the context of production, the mixing of the constituents of the aqueous solution can be effected by stirring, preferred periods for mixing are 2 to 20 h. Before ¬ given to work in the dark, to avoid a Lichtinduz ierte oxidation.
Ein weiterer Gegenstand der Erfindung ist ein Feststoff zur Verwendung als Medikament bei der Behandlung von malignem Melanom, der erfindungsgemäß erhältlich ist durch Entfernen des Lösungsmittels aus einer oben beschriebenen erfindungs¬ gemäßen wässrigen Lösung. Das Entfernen kann bevorzugt durch Gefriertrocknung (Lyophilisation) erfolgen. Sowohl die erfindungsgemäße wässrige medikamentöse Lösung als auch der medikamentöse Feststoff besitzen eine hohe Lagerstabi¬ lität. Another object of the invention is a solid for use as a medicament in the treatment of malignant melanoma, which is obtainable according to the invention by removing the solvent from an inventive aqueous solution described above ¬ . The removal can preferably be carried out by freeze-drying (lyophilization). Both the aqueous drug solution of the invention and the drug solids have high storage stability ¬ formality.
Die Erfindung soll nun im Folgenden weiter in den Beispielen unter Bezug auf die beigefügten Figuren beschrieben werden, ohne auf diese beschränkt zu sein. The invention will now be described further below in the examples with reference to the attached figures, without being limited thereto.
Figur 1 zeigt Histogramme von mit Delphinidin-SBE-ß-CD behandelten (b) und unbehandelten (a) Zellen der humanen Melanom-Zelllinie A-375.
zeigt die mittels sub-Gl-Peak-Methode ermittel¬ te dosisabhängige Apoptoseinduktion durch Figure 1 shows histograms of delphinidin-SBE-β-CD treated (b) and untreated (a) cells of the human melanoma cell line A-375. shows the dose-dependent apoptosis induction determined by means of the sub-Gl peak method
Delphinidin-SBE-ß-CD und Delphinidin Cl im Vergleich zu den Kontrollen SBE-ß-CD, DMSO sowie unbehandelten A-375 Zellen bei niedriger Zell- konfluenz . zeigt die mittels sub-Gl-Peak-Methode ermittel¬ te dosisabhängige Apoptoseinduktion von Delphinidin-SBE-ß-CD im Vergleich zu Delphinidin Cl und den Kontrollen SBE-ß-CD, DMSO sowie unbehandelten A-375 Zellen bei hoher Zellkonfluenz . zeigt die mittels WST-l-Assays ermittelte do¬ sisabhängige Zellviabilitätswirkung von Delphinidin-SBE-ß-CD und Delphinidin Cl im Vergleich zu den Kontrollen SBE-ß-CD, DMSO sowie unbehandelten A-375 Zellen bei niedriger Zellkonfluenz . zeigt die mittels WST-l-Assays ermittelte do¬ sisabhängige Zellviabilitätswirkung von Delphinidin-SBE-ß-CD im Vergleich zu Delphinidin Cl und den Kontrollen SBE-ß-CD, DMSO sowie unbehandelten A-375 Zellen bei hoher Zellkonfluenz . zeigt mikroskopische Aufnahmen von unbehandel¬ ten und mit Delphinidin-SBE-ß-CD, Delphinidin Cl sowie SBE-ß-CD behandelten A-375 Zellen. Delphinidin-SBE-β-CD and delphinidin Cl compared to controls SBE-β-CD, DMSO and untreated A-375 cells with low cell confluency. shows the ermittel by means of sub-Gl-peak method ¬ te dose-dependent apoptosis induction of delphinidin SBE-ß-CD in comparison to delphinidin Cl and controls SBE-ß-CD, DMSO and untreated A-375 cells at high cell confluency. shows the determined by WST-l assays do ¬ sisabhängige Zellviabilitätswirkung of delphinidin-SBE-ß-CD and delphinidin Cl compared to the controls SBE-ß-CD, DMSO and untreated A-375 cells at low cell confluency. shows the determined by WST-l assays do ¬ sisabhängige Zellviabilitätswirkung of delphinidin-SBE-ß-CD compared to delphinidin Cl and the controls SBE-ß-CD, DMSO and untreated A-375 cells at high cell concurrence. shows micrographs of non-commercially ¬ th and delphinidin SBE-ß-CD, delphinidin Cl and SBE-ß-CD-treated A-375 cells.
Figur 5 zeigt Zellzahl und Zellproliferation (Echtzeit- Zell-Monitoring) von mit Delphinidin-SBE-ß-CD behandelten A-375 Zellen hoher Zellkonfluenz im
Vergleich zu unbehandelten Zellen mit dem System XCELLLigence . FIG. 5 shows cell number and cell proliferation (real-time cell monitoring) of delphinidin-SBE-β-CD-treated A-375 cells of high cell confluency in FIG Comparison to untreated cells with the XCELLLigence system.
Figur 6 zeigt Zellzahl und Zellproliferation (Echtzeit- Zell-Monitoring) von mit Delphinidin-SBE-ß-CD behandelten A-375 Zellen niedriger Zell- konfluenz im Vergleich zu unbehandelten Zellen mit dem System XCELLLigence. FIG. 6 shows the cell count and cell proliferation (real-time cell monitoring) of delphinidin-SBE-β-CD-treated A-375 cells of low cell confluency compared to untreated cells with the XCELLLigence system.
Figur 7 zeigt Zellzahl und Zellproliferation (Echtzeit- Zell-Monitoring) von mit Delphinidin Cl behandelten A-375 Zellen hoher Zellkonfluenz im Vergleich zu unbehandelten Zellen mit dem System XCELLLigence . FIG. 7 shows cell counts and cell proliferation (real-time cell monitoring) of delphinidin Cl-treated A-375 cells of high cell confluence compared to untreated cells with the system XCELLLigence.
Figur 8 zeigt Zellzahl und Zellproliferation (Echtzeit- Zell-Monitoring) von mit Delphinidin Cl behandelten A-375 Zellen niedriger Zellkonfluenz im Vergleich zu unbehandelten Zellen mit dem System XCELLLigence. FIG. 8 shows cell counts and cell proliferation (real-time cell monitoring) of delphinidin Cl-treated A-375 cells with low cell confluence compared to untreated cells with the XCELLLigence system.
Figur 9 zeigt Zellzahl und Zellproliferation (Echtzeit- Zell-Monitoring) von mit SBE-ß-CD behandelten A-375 Zellen hoher Zellkonfluenz im Vergleich zu unbehandelten Zellen mit dem System XCELLLigence . FIG. 9 shows the cell count and cell proliferation (real-time cell monitoring) of SBE-β-CD-treated A-375 cells of high cell confluence compared to untreated cells with the XCELLLigence system.
Figur 10 zeigt Zellzahl und Zellproliferation (Echtzeit- Zell-Monitoring) von mit SBE-ß-CD behandelten A-375 Zellen niedriger Zellkonfluenz im Vergleich zu unbehandelten Zellen mit dem System XCELLLigence .
Figur IIa zeigt die mittels sub-Gl-Peak-Methode ermittel¬ te Apoptoseinduktion durch Delphinidin-SBE-ß-CD mit und ohne TRAIL im Vergleich zu den Kontrol¬ len (unbehandelte Zellen bzw. nur mit TRAIL behandelte Zellen) bei niedriger Zellkonfluenz . FIG. 10 shows the cell count and cell proliferation (real-time cell monitoring) of SBE-β-CD-treated A-375 cells of low cell confluence compared to untreated cells with the XCELLLigence system. FIG IIa shows the ermittel by means of sub-Gl-peak method ¬ te apoptosis induction by delphinidin SBE-ß-CD and without TRAIL compared to the Kontrol ¬ len (untreated cells or only with TRAIL-treated cells) at low cell confluency.
Figur IIb zeigt die mittels sub-Gl-Peak-Methode ermittel¬ te Apoptoseinduktion durch Delphinidin-SBE-ß-CD mit und ohne TRAIL im Vergleich zu den Kontrol¬ len (unbehandelte Zellen bzw. nur mit TRAIL behandelte Zellen, mit SBE-ß-CD behandelte Zel¬ len) bei hoher Zellkonfluenz . FIG IIb shows the ermittel by means of sub-Gl-peak method ¬ te apoptosis induction by delphinidin SBE-ß-CD with or without TRAIL compared to the Kontrol ¬ len (untreated cells or only with TRAIL, cells treated with SBE-ß -CD treated cell h ¬ len) at high cell confluency.
Figur 12a zeigt die mittels WST-l-Assays ermittelte Zell- viabilitätswirkung von Delphinidin-SBE-ß-CD mit TRAIL im Vergleich zu Delphinidin-SBE-ß-CD allein und den Kontrollen (unbehandelte Zellen bzw. nur mit TRAIL behandelte Zellen) bei nied¬ riger Zellkonfluenz . FIG. 12a shows the cell viability effect of delphinidin-SBE-β-CD with TRAIL as determined by WST-1 assays in comparison with delphinidin-SBE-β-CD alone and the controls (untreated cells or TRAIL-treated cells only) nied ¬ engined cell confluence.
Figur 12b zeigt die mittels WST-l-Assays ermittelte Zell- viabilitätswirkung von Delphinidin-SBE-ß-CD mit TRAIL im Vergleich zu Delphinidin-SBE-ß-CD allein und den Kontrollen (unbehandelte Zellen bzw. nur mit TRAIL behandelte Zellen, mit SBE- ß-CD behandelte Zellen) bei hoher Zell¬ konfluenz . FIG. 12b shows the cell viability effect of delphinidin-SBE-β-CD with TRAIL as determined by WST-1 assays in comparison with the delphinidin-SBE-β-CD alone and the controls (untreated cells or TRAIL-treated cells only) SBE-ß-CD-treated cells) at high cell ¬ confluence.
Figur 13 zeigt mikroskopische Aufnahmen von unbehandel¬ ten, nur mit TRAIL behandelten, nur mit Delphinidin-SBE-ß-CD behandelten, sowie mit Delphinidin-SBE-ß-CD und TRAIL behandelten A-375 Zellen
Figur 14 zeigt Zellzahl und Zellproliferation (Echtzeit- Zell-Monitoring) von mit TRAIL, Delphinidin- SBE-ß-CD, TRAIL und Delphinidin-SBE- ß-CD behandelten A-375 Zellen hoher Zellkonfluenz im Vergleich zu unbehandelten Zellen mit dem System XCELLLigence . 13 shows micrographs of non-commercially ¬ ten, treated with TRAIL, treated only with delphinidin SBE-ß-CD, and treated with delphinidin SBE-ß-CD and TRAIL A-375 cells FIG. 14 shows the cell count and cell proliferation (real-time cell monitoring) of A-375 cells of high cell confluence treated with TRAIL, delphinidin-SBE-β-CD, TRAIL and delphinidin-SBE-β-CD in comparison with untreated cells with the XCELLLigence system ,
Figur 15 zeigt Zellzahl und Zellproliferation (Echtzeit- Zell-Monitoring) von mit TRAIL, Delphinidin Cl, TRAIL und Delphinidin Cl behandelten A-375 Zellen hoher Zellkonfluenz im Vergleich zu unbehandelten Zellen mit dem System XCELLLigence. Figure 15 shows cell counts and cell proliferation (real-time cell monitoring) of high cell confluency A-375 cells treated with TRAIL, delphinidin Cl, TRAIL and delphinidin Cl compared to untreated cells with the XCELLLigence system.
Figur 16 zeigt Zellzahl und Zellproliferation (Echtzeit- Zell-Monitoring) von mit TRAIL (0,02 pg/ml), SBE-ß-CD (30 pg/ml), TRAIL (0,02 pg/ml) und SBE- ß-CD (30 pg/ml) behandelten A-375 Zellen hoher Zellkonfluenz im Vergleich zu unbehandelten Zellen mit dem System XCELLLigence. FIG. 16 shows cell number and cell proliferation (real-time cell monitoring) of TRAIL (0.02 pg / ml), SBE-β-CD (30 pg / ml), TRAIL (0.02 pg / ml) and SBE-β -CD (30 pg / ml) treated high cell confluency A-375 cells compared to untreated cells with the XCELLLigence system.
Figur 17 zeigt Zellzahl und Zellproliferation (Echtzeit- Zell-Monitoring) von mit TRAIL (0,02 pg/ml), SBE-ß-CD (1000 pg/ml), TRAIL (0,02 pg/ml) und SBE-ß-CD (1000 pg/ml) behandelten A-375 Zellen hoher Zellkonfluenz im Vergleich zu unbehandelten Zellen mit dem System XCELLLigence. Figure 17 shows cell number and cell proliferation (real-time cell monitoring) of TRAIL (0.02 pg / ml), SBE-β-CD (1000 pg / ml), TRAIL (0.02 pg / ml) and SBE-β -CD (1000 pg / ml) treated high cell confluency A-375 cells compared to untreated cells with the XCELLLigence system.
Figur 18 zeigt die Wirkung von DelphinidinCl und des Figure 18 shows the effect of delphinidinCl and the
Delphinidin-SBE-ß-CD Komplexes in Konzentratio¬ nen von 0,1 μΜ, 3,2 μΜ und 100 μΜ im Vergleich zu wirkstofffreiem Kontrollmedium auf die Vitalität von Endothelzellen unter Anwendung des ATP-Lumineszenz-Assays .
Figur 19 zeigt die Wirkung von DelphinidinCl und des Delphinidin-SBE-ß-CD complex in concen ¬ NEN of 0.1 μΜ, 3.2 μΜ and 100 μΜ in comparison to drug-free control medium on the viability of endothelial cells using the ATP luminescence assay. Figure 19 shows the effect of delphinidinCl and the
Delphinidin-SBE-ß-CD Komplexes in Konzentratio¬ nen von 0,1 μΜ, 3,2 μΜ und 100 μΜ im Vergleich zu wirkstofffreiem Kontrollmedium auf die Vitalität von humanen Fibroblasten unter Anwendung des ATP-Lumineszenz-Assays . Delphinidin-SBE-ß-CD complex in concen ¬ NEN of 0.1 μΜ, 3.2 μΜ and 100 μΜ in comparison to drug-free control medium on the vitality of human fibroblasts using the ATP luminescence assay.
Beispiele Examples
I . Herstellung eines Komplexes aus Delphinidin und Cyclo- dextrinen I. Preparation of a complex of delphinidin and cyclodextrins
1. Eingesetzte Materialien: 1. Materials used:
Die nachfolgenden Cyclodextrine werden verwendet: The following cyclodextrins are used:
Delphinidinchlorid wurde von der Firma Extrasynthese erworben. Delphinidin chloride was purchased from the extrasynthesis company.
2. Bestimmung des Delphinidingehalts 2. Determination of delphinidin content
Zur Bestimmung des Gehalts von Delphinidinchlorid in den delphinidinhalt igen Zusammensetzungen wurde ein Reversephase HPLC-Verfahren verwendet. Dabei wurden folgende Rea¬ genzien eingesetzt: To determine the level of delphinidin chloride in the delphinidinhaltigen compositions, a reverse phase HPLC method was used. The following Rea ¬ genzien were used:
Gereinigtes Wasser
Methanol für die Chromatographie Purified water Methanol for chromatography
Ameisensäure, p. a. Formic acid, p. a.
1 M Salzsäure als volumetrische Lösung. 1 M hydrochloric acid as a volumetric solution.
Als Säule wurde eine Waters X Bridge™ C18, 35 μΐ, 150 mm x 4,6 mm verwendet. The column used was a Waters X Bridge ™ C18, 35 μΐ, 150 mm x 4.6 mm.
Die mobilen Phasen waren wie folgt: The mobile phases were as follows:
Kanal A: Wasser 950 ml, Methanol 50 ml, Ameisensäure 10 ml Kanal B: Wasser 50 ml, Methanol 950 ml, Ameisensäure 10 ml Channel A: water 950 ml, methanol 50 ml, formic acid 10 ml Channel B: water 50 ml, methanol 950 ml, formic acid 10 ml
Folgendes Gradientenprogramm wurde verwendet: The following gradient program was used:
Stoppzeit: 35 min Stop time: 35 min
Nachlaufzeit (posttime) : 8 min Follow-up time (post-time): 8 min
Flussrate: 1 ml/min Flow rate: 1 ml / min
In ektionsvolumen: 20 μΐ In ektionsvolumen: 20 μΐ
Säulentemperatur: 30°C +/- 2°C Column temperature: 30 ° C +/- 2 ° C
UV-Vis Detektor: 530 pm für den Assay, 275 pm zur Detektion von Verunreinigungen UV-Vis detector: 530 pm for the assay, 275 pm for the detection of contaminants
Integrator: Fläche Integrator: area
Lösungen und Probenvorbereitung: Solutions and sample preparation:
Verdünnungslösung 1: Mischung aus 100 ml Methanol und 2,6 ml M HCL Dilution solution 1: Mixture of 100 ml of methanol and 2.6 ml of M HCl
Verdünnungslösung 2: Mischung aus 100 ml 40 prozentiger Dilution solution 2: mixture of 100 ml 40 percent
Methanol und 2,6 ml 1 M HCL
Kalibrierungslösung: Eine Referenzlösung von Delphinidin wurde durch Einwiegen von 10 mg Delphinidinchlorid in einen 10 ml Kolben und Lösen in Verdünnungslösung 1 hergestellt. Nach dem Lösen wurde ungefähr lOfach verdünnt mit Verdünnungslösung 2 zur Herstellung einer ungefähren Konzentration von 0,1 mg/ml. Methanol and 2.6 ml 1 M HCL Calibration solution: A reference solution of delphinidin was prepared by weighing 10 mg delphinidin chloride into a 10 ml flask and dissolving in dilution solution 1. After dissolution, it was diluted approximately 10-fold with dilution solution 2 to make an approximate concentration of 0.1 mg / ml.
Die Kontrollkalibrierungslosung wurde auf die gleiche Art und Weise hergestellt. Die Kalibrierungslösungen wurden sofort mittels HPLC analysiert, da Delphinidinchlorid in Lö¬ sung instabil ist. The control calibration solution was prepared in the same manner. The calibration solutions were analyzed immediately by HPLC, since delphinidin chloride is unstable in solution.
Herstellung der Prüflösungen : Preparation of the test solutions:
Zur Bestimmung des Delphinidingehalts erfindungsgemäß her¬ gestellter Feststoffe (Herstellung siehe weiter unten) wurden etwa 50 mg dieser Zusammensetzung in einem 10 ml Kolben eingewogen. Anschließend wurde in Verdünnungslösung 2 gelöst und mit der gleichen Verdünnungslösung 2 weiter verdünnt bis zur Einstellung einer ungefähren Delphinidinkon- zentration von 0,1 mg/ml. To determine the delphinidine content of solids prepared according to the invention (preparation, see below), about 50 mg of this composition were weighed in a 10 ml flask. It was then dissolved in dilution solution 2 and further diluted with the same dilution solution 2 until an approximate delphinidin concentration of 0.1 mg / ml was established.
Die Bestimmung des Delphinidingehalts in den Proben wurde berechnet unter Zuhilfenahme der Agilent ChemStation Soft¬ ware unter Verwendung der Kalibrierung mit dem beschriebenen externen Standard. The determination of the Delphinidingehalts in the samples was calculated with the aid of the Agilent ChemStation software Soft ¬ using the calibration described with the external standard.
Beispiel 1: Komplexierung von Delphinidin mit SBE-ß-CD Example 1 Complexation of Delphinidin with SBE-β-CD
In diesem Beispiel wird die Komplexierung von Delphinidin durch verschiedene Cyclodextrine und die Löslichkeit des Komplexes in wässriger Lösung untersucht.
Es wurden neutrale wässrige Lösungen hergestellt, die 10 Gew.-% des jeweiligen Cyclodextrins enthalten. Bei ß-CD wurde aufgrund der mangelnden Löslichkeit eine Konzentrati¬ on von lediglich 2 Gew.-% gewählt. In this example, the complexation of delphinidin by various cyclodextrins and the solubility of the complex in aqueous solution is investigated. Neutral aqueous solutions containing 10% by weight of the particular cyclodextrin were prepared. When ß-CD due to the lack of solubility of a concentra ¬ one was chosen of only 2 wt .-%.
Je 5ml der wässrigen Cyclodextrinlösungen und von reinem Wasser wurden in Glaskolben gefüllt. Anschließend wurde ein Überschuss Delphinidinchlorid zugegeben. Die erforderliche Überschussmenge war 10 mg für die Lösungen von CC-, ß- und γ-Cyclodextrin und 15 mg für die Lösungen von HPBCD (2- Hydroxypropyl-ß-Cyclodextrin) und SBE-ß-CD. 5 ml each of the aqueous cyclodextrin solutions and of pure water were filled into glass flasks. Subsequently, an excess of delphinidin chloride was added. The required excess amount was 10 mg for the solutions of CC-, β- and γ-cyclodextrin and 15 mg for the solutions of HPBCD (2-hydroxypropyl-β-cyclodextrin) and SBE-β-CD.
Die Suspensionen wurden für 20 h bei 30° C im Dunkeln gerührt. Anschließend wurde filtriert durch einen Membranfil¬ ter mit 0,22 pm Porengröße. The suspensions were stirred for 20 h at 30 ° C in the dark. The mixture was then filtered through a Membranfil ¬ ter of 0.22 pm pore size.
Die erzielbaren Löslichkeiten sind in der nachfolgenden Tabelle wiedergegeben. The achievable solubilities are given in the table below.
Man erkennt, dass die Komplexierung und die dadurch bewirkte Löslichkeit serhöhung für SBE-ß-CD weit besser ist als für die anderen Cyclodextrine . It can be seen that the complexation and the resulting solubility increase is far better for SBE-β-CD than for the other cyclodextrins.
Beispiel 2: Einfluss des pH-Wertes
In diesem Beispiel wurde der Einfluss des pH-Wertes auf die Löslichkeit eines Delphinidin-SBE-ß-CD in wässriger Lösung untersucht. Nach der Vorschrift von Beispiel 1 wurden wäss- rige Lösungen von SEB-ß-CD hergestellt, diese Lösungen jedoch mit 1 M HCL auf die in Tabelle 2 genannten sauren pH- Werte eingestellt. Anschließend wurde Delphinidinchlorid nach der Vorschrift des Beispiels 1 zugegeben und weiterge¬ arbeitet, als einzige Abweichung wurde die Rührzeit auf 2,5 h begrenzt. Die Ergebnisse sind in der nachfolgenden Tabel¬ le wiedergegeben. Example 2: Influence of the pH In this example, the influence of pH on the solubility of a delphinidin-SBE-β-CD in aqueous solution was investigated. Following the instructions of Example 1, aqueous solutions of SEB-β-CD were prepared, but these solutions were adjusted to the acidic pH values indicated in Table 2 using 1 M HCl. Subsequently, delphinidin chloride was added according to the instructions of Example 1 and weiterge ¬ works, the only deviation, the stirring time was limited to 2.5 h. The results are reproduced in the following Tabel ¬ le.
Man erkennt, dass bei pH-Werten zwischen 4 und 5 die Lös¬ lichkeit des komplexierten Delphinidinchlorids sich etwa um den Faktor 3 gegenüber einem neutralen pH-Wert erhöht. It is evident that at pH values between 4 and 5, the Lös ¬ friendliness of the complexed Delphinidinchlorids increased approximately by a factor of 3 with respect to a neutral pH.
Beispiel 3 Herstellung eines erfindungsgemäßen Feststoffs Example 3 Preparation of a solid according to the invention
In diesem Beispiel wird ein erfindungsgemäßer Komplex als Feststoff formuliert. Zu Vergleichszwecken werden ein In this example, a complex of the invention is formulated as a solid. For comparison purposes, a
Delphinidin/HPBCD Komplex sowie eine Delphini- din/Stärkeformulierung als Feststoff zubereitet. Delphinidin / HPBCD complex and a delphinidin / starch formulation prepared as a solid.
Beispiel 3.1: Delphinidin-SBE-ß-CD Example 3.1: Delphinidin-SBE-β-CD
5 g SEB-ß-CD wurden in 40 ml destilliertem Wasser zu einer klaren Lösung gelöst. Der pH-Wert der Lösung wurde mittels 1 M HCL auf 4,8 eingestellt. Anschließend wurden 0,11 g
Delphinidinchlorid hinzugefügt und 2 h bei 27°C im Dunkeln gerührt. Die homogene Flüssigkeit wurde durch einen Cellu- losenitratmembranfilter mit einer Porengröße von 0,45 pm vakuumfiltriert. Die Lösung wurde gefroren und anschließend gefriergetrocknet bei -48°C und einem Druck von etwa 10,3 Pa (77 mTorr) . Das Lyophilisat wurde gemahlen und durch ein Sieb von 0,3 mm Maschenweite gesiebt. 5 g of SEB-β-CD were dissolved in 40 ml of distilled water to a clear solution. The pH of the solution was adjusted to 4.8 by 1 M HCl. Subsequently, 0.11 g Delphinidinchlorid added and stirred for 2 h at 27 ° C in the dark. The homogeneous liquid was vacuum filtered through a 0.45 μm pore size cellulose nitrate membrane filter. The solution was frozen and then freeze-dried at -48 ° C and a pressure of about 10.3 Pa (77 mTorr). The lyophilizate was ground and sieved through a sieve of 0.3 mm mesh size.
Beispiel 3.2: Delphinidin HPBCD Example 3.2: Delphinidin HPBCD
Es wurde in gleicher Weise wie Beispiel 3.1 gearbeitet, je¬ doch wurde bei der Filtration eine signifikante Menge Mate¬ rial abfiltriert, was darauf hindeutet, dass die Solubili- sierung deutlich weniger effektiv war als bei Verwendung von SBE-ß-CD gemäß Beispiel 3.1. It was worked in the same manner as Example 3.1, but ¬ a significant amount of material was filtered off ¬ filtration, suggesting that the solubilization was significantly less effective than when using SBE-ß-CD according to Example 3.1 ,
Beispiel 3.3 Delphinidin Stärkeformulierung Example 3.3 Delphinidin Starch Formulation
5 g Stärke wurde in 40 ml destilliertem Wasser suspendiert. Man erhielt eine weiße Suspension. Der pH-Wert der Lösung wurde mit 1 M HCL auf 4,6 eingestellt. Anschließend wurde 0,11 g Delphinidinchlorid zugegeben und für 2 h bei 27° C im Dunkeln gerührt. Die erhaltene homogene Flüssigkeit wur¬ de wie in Beispiel 3.1 gefriergetrocknet, gemahlen und ge¬ siebt . 5 g of starch was suspended in 40 ml of distilled water. A white suspension was obtained. The pH of the solution was adjusted to 4.6 with 1 M HCl. Subsequently, 0.11 g of delphinidin chloride was added and stirred for 2 h at 27 ° C in the dark. The homogeneous liquid obtained WUR ¬ de freeze-dried as described in Example 3.1, ground and sieved ¬ ge.
Beispiel 3.1 ist erfindungsgemäß, bei den Beispielen 3.2 und 3.3 handelt es sich um Vergleichsbeispiele. Example 3.1 is according to the invention, examples 3.2 and 3.3 are comparative examples.
Beispiel 4: Stabilitätsversuche Example 4: Stability experiments
Die Feststoffe gemäß den Bespielen 3.1 bis 3.3 wurden unter folgenden Bedingungen gelagert:
- 8 Tage bei Raumtemperatur in braunen, verschraubten Glasbehältern, The solids according to Examples 3.1 to 3.3 were stored under the following conditions: - 8 days at room temperature in brown screwed glass containers,
- anschließend 22 Tage bei Raumtemperatur in Glasbehältern unter Sauerstoffatmosphäre im Dunkeln. - Then for 22 days at room temperature in glass containers under an oxygen atmosphere in the dark.
Die letzen 22 Tage der oben beschriebenen Lagerung wurden in Glasviolen mit einem Volumen von 20 ml durchgeführt. Jeweils 250 mg der vorher bereits für 8 Tage gelagerten Pro¬ ben wurden dort eingefüllt, die Violen wurden mit einem Gummistopfen verschlossen und abgedichtet. Mittels zwei Injektionsnadeln wurde der Kopfraum der Violen mit reinem Sauerstoff gespült. Die Proben wurden anschließend im Dun¬ keln gelagert. The last 22 days of the storage described above were carried out in glass vials with a volume of 20 ml. 250 mg each of the previously already stored for 8 days Pro ¬ ben were charged there, the vials were sealed with a rubber stopper and sealed. Using two hypodermic needles, the headroom of the violas was flushed with pure oxygen. The samples were then stored in Dun ¬ angles.
Der Delphinidingehalt der Feststoffe (berechnet als Delphi- nidinchlorid und angegeben in Gew.-%) wurde mittels der oben beschriebenen HPLC-Methode bestimmt. Die Ergebnisse finden sich in der nachfolgenden Tabelle. The delphinidine content of the solids (calculated as dolphinidinium chloride and reported in wt%) was determined by the HPLC method described above. The results can be found in the following table.
Die Ergebnisse zeigen, dass sich erfindungsgemäß ein Delpi- nidinkomplex herstellen lässt, der selbst unter reiner Sau¬ erstoffatmosphäre eine hohe Stabilität und damit gute La¬ gerfähigkeit besitzt. Der Komplex besitzt ferner eine gute Löslichkeit in wässrigen, insbesondere leicht sauren Lösun¬ gen, sodass sich Delphinidin erfindungsgemäß auf vielfälti¬ ge Art und Weise formulieren lässt. Die Stabilität des er¬ findungsgemäßen Feststoffes ist ähnlich gut wie eine Formu-
lierung mit Stärke (Beispiel 3.3), dieses Vergleichsbei¬ spiel lässt sich allerdings nicht in einer wässrigen Lösung formulieren . The results show that a Delpi- be produced nidinkomplex invention, which has a high stability and good La ¬ gerfähigkeit even under pure Sau ¬ erstoffatmosphäre. The complex also has a good solubility in aqueous, in particular slightly acidic Lösun ¬ gene, so that delphinidin according to the invention can be formulated to vielfälti ¬ ge manner. The stability of it ¬ inventive solid is as good as a formulated However-regulation with starch (Example 3.3), this comparative example ¬ play can not be formulated in an aqueous solution.
Beispiel 5: Stabilitätsversuche in wässriger Lösung Example 5 Stability Tests in Aqueous Solution
Zur Bestimmung des Gehalts von Delphinidinchlorid in den delphinidinhaltigen Lösungen wurde ein Reverse Phase HPLC- Verfahren ähnlich dem oben bereits beschriebenen verwendet. Dabei wurden folgende Reagenzien eingesetzt: To determine the level of delphinidin chloride in the delphinidinhaltigen solutions a reverse phase HPLC method similar to the one already described above was used. The following reagents were used:
Gereinigtes Wasser Purified water
Methanol für die Chromatographie Methanol for chromatography
Armeisensäure, p. a. Formic acid, p. a.
1 M Salzsäure als volumetrische Lösung. 1 M hydrochloric acid as a volumetric solution.
Als Säule wurde eine Waters X Bridge™ C18, 35 μΐ, 150 mm x 4,6 mm verwendet. The column used was a Waters X Bridge ™ C18, 35 μΐ, 150 mm x 4.6 mm.
Die mobilen Phasen waren wie folgt: The mobile phases were as follows:
Kanal A: Wasser 770 ml, Methanol 230 ml, Armeisensäure 10 ml Kanal B: Wasser 50 ml, Methanol 950 ml, Armeisensäure 10 ml Channel A: water 770 ml, methanol 230 ml, formic acid 10 ml Channel B: water 50 ml, methanol 950 ml, formic acid 10 ml
Folgendes Gradientenprogramm wurde verwendet: The following gradient program was used:
Stoppzeit: 25 min Stop time: 25 min
Nachlaufzeit (posttime) : 8 min
Flussrate: 1 ml/min Follow-up time (post-time): 8 min Flow rate: 1 ml / min
In ektionsvolumen: 20 μΐ In ektionsvolumen: 20 μΐ
Säulentemperatur: 30°C +/- 2°C Column temperature: 30 ° C +/- 2 ° C
UV-Vis Detektor: 530 pm für den Assay, 275 pm zur Detektion von Verunreinigungen UV-Vis detector: 530 pm for the assay, 275 pm for the detection of contaminants
Integrator: Fläche Integrator: area
Lösungen und Probenvorbereitung: Solutions and sample preparation:
Verdünnungslösung 1: Mischung aus 100 ml Methanol und 2,6 ml 1 M HCL Dilution solution 1: Mixture of 100 ml of methanol and 2.6 ml of 1 M HCl
Verdünnungslösung 2: Mischung aus 100 ml 50 %igem Dilution solution 2: Mixture of 100 ml 50%
Methanol und 2,6 ml 1 M HCL Methanol and 2.6 ml 1 M HCL
Kalibrierungslösung: Eine Referenzlösung von Delphinidin wurde durch Einwiegen von 10 mg Delphinidinchlorid in einen 10 ml Kolben und Lösen in Verdünnungslösung 1 hergestellt. Nach dem Lösen wurde ungefähr lOfach verdünnt mit Verdünnungslösung 2 zur Herstellung einer ungefähren Konzentration von 0,1 mg/ml. Calibration solution: A reference solution of delphinidin was prepared by weighing 10 mg delphinidin chloride into a 10 ml flask and dissolving in dilution solution 1. After dissolution, it was diluted approximately 10-fold with dilution solution 2 to make an approximate concentration of 0.1 mg / ml.
Die Kontrollkalibrierungslösung wurde auf die gleiche Art und Weise hergestellt. Die Kalibrierungslösungen wurden sofort mittels HPLC analysiert, da Delphinidinchlorid in Lö¬ sung instabil ist. The control calibration solution was prepared in the same manner. The calibration solutions were analyzed immediately by HPLC, since delphinidin chloride is unstable in solution.
Herstellung der Prüflösungen : Preparation of the test solutions:
Zur Bestimmung des Delphinidingehalts einer erfindungsgemä¬ ßen wässrigen Lösung wurden Delphinidin/SBE-ß-CD des Beispiels 3.1 (erfindungsgemäß) und Delphinidin (Vergleichs-
beispiel in 0,9 %iger NaCl-Lösung gelöst bis zur Einstel¬ lung einer Startkonzentration (bezogen auf das Delphinidin) von 1,584 mg/ml (erfindungsgemäßes Beispiel) bzw. 0,0216 mg/ml (Vergleichsbeispiel). Die Lösungen wurden bei Raum¬ temperatur hergestellt und anschließend bei 37°C im Dunkeln in verschlossenen Violen gelagert. To determine the Delphinidingehalts an inventive ¬ SEN aqueous solution delphinidin / SBE-ß-CD of Example 3.1 (Invention) and delphinidin have been (comparative for example in 0.9% NaCl solution dissolved to SET lung ¬ a starting concentration (based on the delphinidin) of 1.584 mg / ml (Inventive example) and 0.0216 mg / ml (Comparative Example). The solutions were prepared at room temperature ¬ and then stored at 37 ° C in the dark in sealed vials.
Nach 1, 2, 3 und 4 h wurde der Delphinidingehalt bestimmt. Die nachfolgende Tabelle gibt den ermittelten Gehalt als Prozentanteil der oben genannten Startkonzentration an. After 1, 2, 3 and 4 h, the delphinidine content was determined. The following table indicates the determined content as a percentage of the above-mentioned starting concentration.
Die Bestimmung des Delphinidingehalts in den Proben wurde berechnet unter Zuhilfenahme der Agilent ChemStation Soft¬ ware unter Verwendung der Kalibrierung mit den beschriebenen externen Standard. The determination of the Delphinidingehalts in the samples was calculated with the aid of the Agilent ChemStation software Soft ¬ using the calibration with the described external standard.
II. Wirkung von Delphinidin und des Delphinidin-SBE-ß-CD Komplexes auf Melanomzellen II. Effect of Delphinidin and the Delphinidin-SBE-β-CD Complex on Melanoma Cells
Test zelllinie Test cell line
In den nachfolgend beschriebenen in-vitro Versuchen wurde die Wirkung des Komplexes aus Delphinidin und Sulfobuty- lether-ß-Cyclodextrin (nachfolgend Delphinidin-SBE-ß-CD) und Delphinidin auf die exemplarisch verwendete humane Me- lanom-Zelllinie A-375 [ATTC Katalog Nr. CRL-1619; Brüggen J., Sorg C. (1983) Detection of phenotypic differences on
human malignant melanoma lines and their variant sublines with monoclonal antibodies. Cancer Immunol Immunother 15: 200-205] untersucht. In the in vitro experiments described below, the effect of the complex of delphinidin and sulfobutyl ether-.beta.-cyclodextrin (hereafter delphinidin-SBE-.beta.-CD) and delphinidin on the exemplary human melanoma cell line A-375 [ATTC Catalog No. CRL-1619; Brüggen J., Sorg C. (1983) Detection of phenotypic differences on human malignant melanoma lines and their variant sublines with monoclonal antibodies. Cancer Immunol Immunother 15: 200-205].
Die Zelllinie wurde bei 37°C, 5% C02 in DMEM (Dulbecco's Modified Eagle's Medium von Gibco, Karlsruhe, Deutschland) ergänzt mit 10% FCS (Fetal Bovine Serum) und Antibiotika kultiviert . The cell line was cultured at 37 ° C, 5% C0 2 in DMEM (Dulbecco's Modified Eagle's medium from Gibco, Karlsruhe, Germany) supplemented with 10% FCS (Fetal Bovine Serum) and antibiotics.
Beispiel 6 Example 6
(Apoptoseinduktionsmessung mit Hilfe der sub-Gi-Peak- (Apoptosis induction measurement using the sub-Gi peak
Methode) Method)
In dem Experiment gemäß Beispiel 6 wurde die Wirkung der untersuchten Substanzen auf die Apoptoseinduktion bei der Testzelllinie mit Hilfe der sub-Gi-Peak-Methode, die auch unter Bezeichnung „Nicoletti-Methode" in der Fachliteratur geläufig ist, untersucht [Riccardi C. Nicoletti I. (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat . Protoc 1: 1458-1461]. In the experiment according to Example 6, the effect of the investigated substances on the induction of apoptosis in the test cell line using the sub-Gi-peak method, which is also known under the name "Nicoletti method" in the literature, studied [Riccardi C. Nicoletti I. (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry, Nat. Protoc 1: 1458-1461].
Die Methode basiert auf dem geringeren DNA-Gehalt apoptoti- scher Zellen im Vergleich zu vitalen Zellen. Ein Merkmal der Apoptose ist die Spaltung der DNA durch Endonukleasen in kurze DNA-Fragmente, wodurch in apoptotischen Zellen im Vergleich zu vitalen Zellen der Gehalt an niedrigmolekularer DNA erhöht und der Anteil an hochmolekularer DNA reduziert ist. Bei induzierter Lyse (Permeabilisierung) der Zellmembran treten die niedermolekularen DNA Fragmente aus den apoptotischen Zellen aus. Werden die Zellen mit Propi- diumiodid (PI), einem in die DNA-interkalierenden Farb¬ stoff, gefärbt, fluoreszieren apoptotische Zellen weniger intensiv als vitale Zellen und der durchflusszytometrisch bestimmte DNA-Gehalt erscheint bei apoptotischen Zellkernen als breiter, hypodiploider , schwächer-fluoreszierender DNA- Peak ( sub-Gi-Peak ) , der leicht vom schmalen DNA-Doppelpeak gesunder Zellen mit diploidem DNA-Gehalt unterschieden werden kann. Ein Beispiel dafür ist mit den in Figur 1 dargestellten Histogrammen a und b gegeben. Histogramm a zeigt
unbehandelte, PI-gefärbte Zellen. Propidiumiodid hat sich in die DNA eingelagert und fluoresziert in zwei schmalen Peaks, die Zellen der Gl-Phase (linker Peak) mit einfachem und Zellen in der G2-Phase (rechter Peak) mit doppeltem DNA-Gehalt. Ein dem linken Peak vorgelagerter sub-Gi-Peak fehlt nahezu. Histogramm b zeigt dagegen einen breiten sub- Gi-Peak. Dies stellt den Anteil apoptotischer Zellkerne nach 24 Stunden Delphinidin-SBE-ß-CD-Behandlung dar. The method is based on the lower DNA content of apoptotic cells compared to vital cells. A feature of apoptosis is the cleavage of the DNA by endonucleases into short DNA fragments, which increases the content of low molecular weight DNA in apoptotic cells compared to vital cells and reduces the proportion of high molecular weight DNA. Upon induced lysis (permeabilization) of the cell membrane, the low molecular weight DNA fragments escape from the apoptotic cells. If the cells diumiodid with propionaldehyde (PI), a in the DNA intercalating color ¬ material, colored, fluorescent apoptotic cells less intense appears as vital cells and flow cytometry certain DNA content in apoptotic nuclei as a broad, hypodiploid, weaker-fluorescent DNA peak (sub-Gi peak), which can be easily distinguished from the narrow DNA double peak of healthy cells with diploid DNA content. An example of this is given with the histograms a and b shown in FIG. Histogram a shows untreated, PI-stained cells. Propidium iodide has been incorporated into the DNA and fluoresces in two narrow peaks, the Gl phase (left peak) with single and the cells in the G2 phase (right peak) with double DNA content. A sub-Gi peak upstream of the left peak is nearly absent. Histogram b, on the other hand, shows a broad subGi peak. This represents the proportion of apoptotic cell nuclei after 24 hours of delphinidin-SBE-β-CD treatment.
Für die Durchführung der sub-Gl-peak-Methode gemäß Beispiel 6 wurden die Zellen für 24 Stunden mit 10-3000 pg Delphini- din-SBE-ß-CD pro ml Zellsuspension und 15-120 μΜ gereinigtes Delphinidinchlorid (nachfolgend Delphinidin Cl) pro ml Zellsuspension inkubiert, wobei mit dem Komplexpartner SBE- ß-CD sowie DMSO behandelte Zellen, als auch unbehandelte Zellen als Kontrollen dienten. Für jeden Einzelversuch (Kontrolle, Delphinidin-SBE-ß-CD, SBE-ß-CD, Delphinidin Cl) wurden drei Vertiefungen a 3 ml auf einer Well-platte mit Zellen angesetzt. Anschließend wurden die Zellen mittels Trypsinierung geerntet, mit eiskalter Phosphatgepufferter Salzlösung (PBS) gewaschen und für 1 Stunde mit dem Färbungspuffer enthaltend 0,1% Natriumzitrat , 0,1% Triton X- 100 und PI (40pg/ml; Sigma-Aldrich, Taufkirchen, Deutschland) inkubiert, bevor anschließend der DNA-Gehalt der Zellkerne mit dem Durchflusszytometer gemessen und ausgewertet wurde (FACSCalibur und CellQuest Software; Becton Dickinson, Heidelberg) . Aus den jeweils drei Messwerten wurde der Mittelwert berechnet, wobei die Messwerte der Kontrollzellen als Referenz für den t-Test dienten. For carrying out the sub-Gl-peak method according to Example 6, the cells were incubated for 24 hours with 10-3000 pg delphinidin-SBE-β-CD per ml cell suspension and 15-120 μΜ purified delphinidin chloride (hereinafter delphinidin Cl) per incubated with the complex partner SBE-ß-CD and DMSO treated cells, as well as untreated cells as controls. For each individual experiment (control, delphinidin-SBE-.beta.-CD, SBE-.beta.-CD, delphinidin Cl) three wells a 3 ml were placed on a well plate with cells. The cells were then harvested by trypsinization, washed with ice-cold phosphate buffered saline (PBS) and stained for 1 hour with the staining buffer containing 0.1% sodium citrate, 0.1% Triton X-100 and PI (40pg / ml; Sigma-Aldrich, Taufkirchen , Germany) before subsequently measuring and evaluating the DNA content of the cell nuclei with the flow cytometer (FACSCalibur and CellQuest Software, Becton Dickinson, Heidelberg). From each of the three measured values, the mean value was calculated, with the measured values of the control cells serving as a reference for the t-test.
Es ist bekannt, dass bei Apoptosemessungen eine geringe spontane Apoptoserate (Hintergrundapoptose) auftreten kann, wobei dieser Effekt insbesondere dann auftritt, wenn Zellen bereits das Wachstumsplateau erreicht haben und an Nähr¬ stoff- und Sauerstoffmangel leiden. Um dies zu berücksich¬ tigen wurde an Zellen gemessen, die sich in der exponen- tiellen Wachstumsphase befanden, wobei der Versuch parallel sowohl bei geringer (niedrige Zellkonfluenz ) , als auch höherer Zelldichte (höhere Zellkonfluenz ) durchgeführt wurde,
dessen Ergebnisse in den Figuren 2a (niedrige Zell- konfluenz) und 2b (höhere Zellkonfluenz ) graphisch aufbereitet dargestellt sind. It is known that in Apoptosemessungen a low spontaneous Apoptoserate (background apoptosis) may occur, this effect occurs especially when cells have already reached the growth plateau and suffer from nutrient ¬ material and oxygen deficiency. To make this into account ¬ term was measured on cells that were in the exponential growth phase, the test parallel both at low (low cell confluency) as well as higher cell density (higher cell confluence) was performed, the results of which are shown graphically in FIGS. 2a (low cell confluency) and 2b (higher cell concenlacy).
VerSuchsergebnisse test results
- Gereinigtes Delphinidin Cl zeigt dosisabhängig eine sig¬ nifikante Apoptoseinduktion (vgl. Figur 2a bei 120 μΜ) ;- Purified delphinidin Cl shows a dose-dependent sig ¬ nifikante induction of apoptosis (see Figure 2a at 120 μΜ.)
- Eine deutlich stärke Apoptoseinduktion zeigt der Delphi- nidin-SBE-ß-CD-Komplex (vgl. Figur 2a bei 1000 pg/ml und Figur 2b bei 3000 pg/ml); The dolphin-SBE-β-CD complex shows a markedly strong induction of apoptosis (see Figure 2a at 1000 pg / ml and Figure 2b at 3000 pg / ml);
- Während der Komplexpartner SBE-ß-CD sowie DMSO keine Wirkung zeigt, analog den unbehandelten Kontrollzellen. - While the complex partner SBE-ß-CD and DMSO shows no effect, analogous to the untreated control cells.
Beispiel 7 Example 7
(Zellviabilitätstest) (Zellviabilitätstest)
In dem Experiment gemäß Beispiel 7 wurde die Wirkung der untersuchten Substanzen auf die Zellviabilität der Test¬ zelllinie mit Hilfe des WST-l-Assays (Water S_oluble Tetra- zolium) von Roche Diagnostics quantifiziert. Der WST-1- Assay dient zum Nachweis einer intakten Atmungskette in Zellen, wobei viable Zellen mit einem intakten mitochondrialen Succinat-Tetrazolium-Dehydrogenase-System eine enzyma- tische Umsetzung des schwach rot gefärbten In the experiment according to Example 7, the effect of the tested substances on the cell viability of the test ¬ cell line using the WST-I assay (Water S_oluble tetra- zolium) from Roche Diagnostics was quantified. The WST-1 Assay serves to detect an intact respiratory chain in cells, viable cells with an intact mitochondrial succinate-tetrazolium dehydrogenase system an enzymatic reaction of the weakly red colored
Tetrazoliumsalzes WST-1 ( 4- [ 3- ( 4-Iodophenyl ) -2- ( 4- nitrophenyl ) -2H-5-tetrazolio ] -1 , 3-Benzol-Disulfonat ) in das dunkelrote Formazan bewirken. Dieser Farbumschlag kann in einem Spektralphotometer photometrisch gemessen und ausgewertet werden. Tetrazolium salt WST-1 (4- [3- (4-iodophenyl) -2- (4-nitrophenyl) -2H-5-tetrazolio] -1,3-benzene disulfonate) into the dark red formazan. This color change can be photometrically measured and evaluated in a spectrophotometer.
Für die Durchführung des WST-l-Assays gemäß Beispiel 7 wur¬ den die Zellen analog Beispiel 6 für 24 Stunden mit 10-3000 pg Delphinidin-SBE-ß-CD pro ml Zellsuspension bzw. 15-120 μΜ gereinigtes Delphinidin Cl pro ml Zellsuspension inkubiert, wobei mit dem Komplexpartner SBE-ß-CD sowie DMSO be¬ handelte Zellen, als auch unbehandelte Zellen als Kontrol¬ len dienten. An den WST-l-Assay, wie beispielsweise in
Plötz et al . (2012), Mutual Regulation of Bcl-2 Proteins Independent of the BH3 Domain as Shown by the BH3-Lacking Protein Bcl-xAK, PLOS ONE, vol 7, issue 4, e34549 im Einzelnen beschrieben, schlössen sich die Färbung mit Calcein- AM mittels Inkubation mit Calcein ( 4μΜ; e Bioscience, Frankfurt, Deutschland) in Serum-freien Wachstumsmedium für 60 Minuten bei 37°C sowie eine Waschung mit PBS an, bevor die Zellviabilitätsmessung mittels Durchflusszytometrie (s. Beispiel 5) erfolgte um Calcein-gefärbte (lebende) Zellen von ungefärbten (toten) Zellen zu unterscheiden. For the implementation of the WST-I assay as described in Example 7 ¬ WUR which the cells of Example 6 for 24 hours with 10-3000 pg delphinidin SBE-ß-CD per ml of cell suspension or 15-120 μΜ purified delphinidin Cl per ml of cell suspension incubated with the complex partners SBE-ß-CD and DMSO ¬ be acted cells, and untreated cells served as Kontrol ¬ len. To the WST-I assay, such as in Plötz et al. (2012), Mutual Regulation of Bcl-2 Proteins Independently of the BH3 Domain as Shown by the BH3-lacquering protein Bcl-xAK, PLOS ONE, vol 7, issue 4, e34549, staining with calcein AM by incubation with calcein (4 μl, e Bioscience, Frankfurt, Germany) in serum-free growth medium for 60 minutes at 37 ° C. and washing with PBS before the cell viability measurement by flow cytometry (see Example 5) was carried out using calcein-stained ( living) cells from unstained (dead) cells.
Analog Beispiel 6 wurde auch in Beispiel 7 der Versuch so¬ wohl bei geringer (niedrige Zellkonfluenz ) , als auch höherer Zelldichte (höhere Zellkonfluenz ) durchgeführt, dessen Ergebnisse in den Figuren 3a (niedrige Zellkonfluenz ) und 3b (höhere Zellkonfluenz ) graphisch aufbereitet dargestellt sind . Analogously to Example 6, the experiment was also carried out in Example 7 so ¬ probably at low (low cell concurrence), as well as higher cell density (higher cell concurrency), the results in Figures 3a (low cell confluence) and 3b (higher cell concurrence) are presented graphically processed ,
VerSuchsergebnisse test results
- Gereinigtes Delphinidin Cl zeigt mit steigender Dosis ei¬ nen Verlust an vitalen Zellen bei niedriger Zellkonfluenz (vgl . Figur 3a) ; - Purified delphinidin Cl shows with increasing dose ei ¬ nen loss of vital cells at low cell confluence (see Figure 3a);
- Eine deutlich stärkeren und signifikanten Verlust an - A much stronger and significant loss
Zellvitalität bewirkt der Delphinidin-SBE-ß-CD-Komplex (vgl. Figur 3a bei 1000 pg/ml und 2000 pg/ml und Figur 3b bei 3000 pg/ml) ; Cell vitality is effected by the delphinidin-SBE-β-CD complex (see Figure 3a at 1000 pg / ml and 2000 pg / ml and Figure 3b at 3000 pg / ml);
- Während der Komplexpartner SBE-ß-CD sowie DMSO keine Wirkung zeigt, analog den unbehandelten Kontrollzellen. - While the complex partner SBE-ß-CD and DMSO shows no effect, analogous to the untreated control cells.
Der über die Apoptoseinduktion (Beispiel 6) hinausgehende Effekt des Verlustes an vitalen Zellen spiegelt sich auch in der Morphologie der Zellen bei mikroskopischer Betrachtung 24 stündiger Behandlung wieder, die sich bei hohen Konzentrationen von Delphinidin Cl bzw. Delphinidin-SBE-ß- CD abkugeln und/oder ablösen, wie aus dem mikroskopischen Aufnahmen in Figur 4 ersichtlich.
Beispiel 8 The effect of loss of vital cells beyond the induction of apoptosis (Example 6) is also reflected in the morphology of the cells when examined microscopically for 24 hours, which precipitate at high concentrations of delphinidin Cl or delphinidin-SBE-β-CD and / or peel off, as can be seen from the microscopic images in Figure 4. Example 8
[Kontinuierliches Echt zeit-Zell-Monitoring (Real-time cell analysis - RTCA) ] [Continuous real-time cell analysis (RTCA)]
Die Zellzahl und Zellproliferation mit gleicher Wirkstoffuntersuchung analog den Beispielen 6 und 7 wurde in Echtzeit mittels des Systems xCELLLigence (Roche Diagnostics, Mannheim, Deutschland) aufgezeichnet. Das xCELLigence Sys¬ tem ist ein mikroelektronisches Biosensorsystem für zellba¬ sierte, markierungsfreie Analysen, das dynamische Zelldaten in Echtzeit liefert. Die Kulturplatten des xCELLigence Sys¬ tems sind mit Mikroelektroden am Boden jeder Mulde ausgestattet, um Änderungen in der elektrischen Impedanz zu messen. Quantitative Impedanzänderungen korrelieren mit der Anzahl und Stärke der Zellkontakte mit den darunter liegenden Elektroden. The cell count and cell proliferation with the same drug study analogous to Examples 6 and 7 was recorded in real time by means of the system xCELLLigence (Roche Diagnostics, Mannheim, Germany). The xCELLigence Sys ¬ tem is a microelectronic biosensor for zellba ¬ catalyzed, label-free analysis, provides the dynamic cell data in real time. The culture plates of xCELLigence Sys tems ¬ are equipped each well with microelectrodes on the ground, in order to measure changes in electrical impedance. Quantitative impedance changes correlate with the number and strength of cell contacts with the underlying electrodes.
Delphinidin-SBE-ß-CD zeigt in Übereinstimmung mit den hohen Apoptoseraten (Beispiel 6) sowie dem Verlust der Zellvita¬ lität (Beispiel 7) einen vollständigen Einbruch der Delphinidin-SBE-ß-CD shows in accordance with the high rates of apoptosis (Example 6) and the loss of Zellvita ¬ lity (Example 7) a complete collapse of the
Zellproliferation (vgl. Figuren 5 und 6), insbesondere starke antiproliferative Effekte ab einer Konzentration von 1000 pg/ml in Figur 5 und eine vollständige Blockade der Zellproliferation ab 500 pg/ml in Figur 6. Cell proliferation (see Figures 5 and 6), in particular strong antiproliferative effects from a concentration of 1000 pg / ml in Figure 5 and a complete blockade of cell proliferation from 500 pg / ml in Figure 6.
Ähnlich starke antiproliferative Effekte zeigen sich mit Delphinidin Cl bei 60-120 μΜ (vgl. Figuren 7 und 8), während sich erwartungsgemäß die unbehandelten Zellen sowie die DMSO-Kontrolle nahezu neutral verhielten (vgl. Figuren 7-10), d.h. die vorbeschriebenen antiproliferativen Wirkungen sind eineindeutig den untersuchten Wirkstoffen Delphinidin-SBE-ß-CD und Delphinidin Cl zuzuschreiben. Similarly strong antiproliferative effects are seen with delphinidin Cl at 60-120 μΜ (see Figures 7 and 8), while the untreated cells and the DMSO control were expected to be nearly neutral (see Figures 7-10), i. the antiproliferative effects described above are unambiguously attributable to the investigated active substances delphinidin-SBE-β-CD and delphinidin Cl.
III. Wirkung von Delphinidin und des Delphinidin-SBE-ß-CD- Komplexes in Kombination mit TRAIL auf Melanomzellen
Test zelllinie und verwendete Verfahren III. Effect of delphinidin and the delphinidin-SBE-ß-CD complex in combination with TRAIL on melanoma cells Test cell line and used procedures
Bei den Versuchen wurde auf die gleiche Tellinie und Ver¬ fahren wie in vorangehendem Kapitel II verwendet. In the experiments, in the same Tellinie and United go ¬ as in Chapter II preceded used.
Beispiel 9 Example 9
(Apoptoseinduktionsmessung mit Hilfe der sub-Gi-Peak- Methode) (Apoptosis induction measurement using the sub-Gi peak method)
In dem Experiment gemäß Beispiel 9 wurde mittels der der sub-Gi-Peak-Methode die Wirkung des Delphinidin-SBE-ß-CD Komplexes in Kombination mit dem proapoptotischen Todesli- ganden TRAIL untersucht, gegen den die Zelllinie A-375 nur moderate Apoptosesensitivität besitzt. Für die Durchführung der sub-Gl-Peak-Methode gemäß Beispiel 9 wurden die Zellen für 24 Stunden mit 30-1000 pg Delphinidin-SBE-ß-CD pro ml Zellsuspension mit und ohne 0,02 pg TRAIL pro ml Zellsuspension inkubiert, wobei mit dem Komplexpartner SBE-ß-CD behandelte Zellen, als auch unbehandelte Zellen als Kon¬ trollen dienten. Die Versuche wurden parallel sowohl bei geringer (niedrige Zellkonfluenz ) , als auch höherer Zelldichte (höhere Zellkonfluenz ) durchgeführt, deren Ergebnis¬ se in den Figuren IIa (niedrige Zellkonfluenz ) und IIb (höhere Zellkonfluenz ) graphisch aufbereitet dargestellt sind. In the experiment according to Example 9, the effect of the delphinidin-SBE-β-CD complex in combination with the pro-apoptotic death ligand TRAIL, against which the cell line A-375 possesses only moderate apoptosis sensitivity, was investigated by means of the sub-Gi peak method , For carrying out the sub-Gl-peak method according to Example 9, the cells were incubated for 24 hours with 30-1000 pg of delphinidin-SBE-β-CD per ml of cell suspension with and without 0.02 pg of TRAIL per ml of cell suspension the complex partners SBE-ß-CD-treated cells served as controls carried out as ¬ and untreated cells. The experiments were carried out in parallel both at low (low cell confluence), and higher cell density (higher cell concurrency), the result ¬ se in the figures IIa (low cell concurrence) and IIb (higher cell concurrency) are shown graphically processed.
Versuchsergebnisse test results
- Bei der Kombination von Delphinidin-SBE-ß-CD mit TRAIL ist die Apoptoseinduktion gegenüber Delphinidin-SBE-ß-CD allein bei allen untersuchten Delphinidin-SBE-ß-CD- Konzentrationen gesteigert; In the case of the combination of delphinidin-SBE-β-CD with TRAIL, the induction of apoptosis with respect to delphinidin-SBE-β-CD alone is increased at all the delphinidin-SBE-β-CD concentrations investigated;
- Ebenfalls ist eine Steigerung der Apoptoseinduktion von Delphinidin-SBE-ß-CD mit TRAIL gegenüber TRAIL allein bei höheren Delphinidin-SBE-ß-CD-Konzentrationen signifikant gesteigert ; - Also, an increase in the induction of apoptosis of delphinidin-SBE-ß-CD with TRAIL against TRAIL alone at higher delphinidin-SBE-ß-CD concentrations is significantly increased;
- Während der Komplexpartner SBE-ß-CD auf Kontrollniveau liegt .
Beispiel 10 - While the complex partner SBE-ß-CD is at the control level. Example 10
(Zellviabilitätstest) (Zellviabilitätstest)
In dem Experiment gemäß Beispiel 10 wurde analog Beispiel 7 die Zellviabilität der Test zelllinie unersucht, mit dem Un¬ terschied, dass die Zellen der Kombination aus Delphinidin- SBE-ß-CD Komplexe und TRAIL ausgesetzt wurden. In the experiment according to Example 10, the cell viability of the test cell line was unersucht analogously to Example 7, with the Un ¬ terschied that the cells of the combination of Delphinidin- SBE-ß-CD complexes and TRAIL were suspended.
Für die Durchführung des Assays analog Beispiel 7 wurden die Zellen für 24 Stunden mit 30-1000 pg Delphinidin-SBE-ß- CD pro ml Zellsuspension mit und ohne 0,02 pg/ml TRAIL pro ml Zellsuspension inkubiert, wobei dem Komplexpartner SBE- ß-CD behandelte Zellen, als auch unbehandelte Zellen als Kontrollen dienten. To carry out the assay analogously to Example 7, the cells were incubated for 24 hours with 30-1000 pg of delphinidin-SBE-β-CD per ml of cell suspension with and without 0.02 pg / ml TRAIL per ml of cell suspension, the complex partner SBE-ß CD-treated cells, as well as untreated cells served as controls.
Analog Beispiel 9 wurde auch in Beispiel 10 der Versuch so¬ wohl bei geringer (niedrige Zellkonfluenz ) , als auch höherer Zelldichte (höhere Zellkonfluenz ) durchgeführt, dessen Ergebnisse in den Figuren 12a (niedrige Zellkonfluenz ) und 12b (höhere Zellkonfluenz ) graphisch aufbereitet darge¬ stellt sind. Analogously to Example 9, the experiment was so ¬ well performed at low (low cell confluency) as well as higher cell density (higher cell confluency) in Example 10, the results displayed graphically in Figures 12a (low cell confluency) and 12b (higher cell confluency) Darge ¬ are.
VerSuchsergebnisse test results
- Bei der Kombination von Delphinidin-SBE-ß-CD mit TRAIL sinkt die Zellvitalität gegenüber dem Delphinidin-SBE-ß- CD allein bei allen untersuchten Delphinidin-SBE-ß-CD- Konzentrationen; - In the combination of delphinidin-SBE-ß-CD with TRAIL the cell vitality against the delphinidin-SBE-ß-CD alone decreases at all delphinidin-SBE-ß-CD concentrations tested;
- Ebenfalls ist eine Absenkung der Zellvitalität die der Kombination von Delphinidin-SBE-ß-CD mit TRAIL gegenüber TRAIL allein bei höheren Delphinidin-SBE-ß-CD- Konzentrationen ersichtlich; - Also, a decrease in cell vitality that the combination of delphinidin-SBE-ß-CD with TRAIL against TRAIL alone at higher delphinidin-SBE-ß-CD concentrations is apparent;
- Während der Komplexpartner SBE-ß-CD auf Kontrollniveau liegt . - While the complex partner SBE-ß-CD is at the control level.
Der über die Apoptoseinduktion (Beispiel 9) hinausgehende Effekt des Verlustes an vitalen Zellen spiegelt sich auch in der Morphologie der Zellen bei mikroskopischer Betrach-
tung 24 Stunden nach der Behandlung wieder, die sich bei zusätzlicher Anwendung von TRAIL zu Delphinidin-SBE-ß-CD noch stärker abkugeln bzw. ablösen, wie aus dem mikroskopischen Aufnahmen in Figur 13 ersichtlich. The effect of loss of vital cells beyond apoptosis induction (Example 9) is also reflected in the morphology of the cells under microscopic observation. Twenty-four hours after the treatment, which become even more pronounced when the TRAIL is added to delphinidin-SBE-β-CD or detached, as can be seen from the microscopic images in FIG.
Beispiel 11 Example 11
[Kontinuierliches Echt zeit-Zell-Monitoring (Real-time cell analysis - RTCA) ] [Continuous real-time cell analysis (RTCA)]
Die Zellzahl und Zellproliferation mit gleicher Wirkstoffuntersuchung wurde analog den Beispielen 9 und 10 und zusätzlich ergänzt um die Kombination aus Delphinidin Cl mit und ohne TRAIL in Echtzeit mittels des Systems xCELLLigence (Roche Diagnostics, Mannheim, Deutschland) aufgezeichnet. The cell number and cell proliferation with the same active substance investigation were recorded analogously to Examples 9 and 10 and additionally supplemented by the combination of delphinidin Cl with and without TRAIL in real time by means of the system xCELLLigence (Roche Diagnostics, Mannheim, Germany).
Delphinidin-SBE-ß-CD zeigt bei Kombination mit TRAIL in Übereinstimmung mit den gegenüber Delphinidin-SBE-ß-CD allein gesteigerten Apoptoseraten (Beispiel 9) sowie dem stärkeren Verlust der Zellvitalität (Beispiel 10) eine sy¬ nergistische und stärkere Blockierung der Zellproliferation (vgl. Figur 14). Eine stärkere Blockierung der Zellproliferation lasst sich ebenfalls bei der Kombination von Delphinidin Cl mit TRAIL gegenüber TRAIL allein und Delphinidin allein feststellen (vgl. Figur 15) . Keine Verstärkung der TRAIL-Sensitivität war durch SBE-ß-CD zu beobachten (vgl. Figuren 16 und 17) d.h. die vorbeschriebenen antiproliferativen Wirkungsverstärkungen sind eineindeutig den untersuchten Wirkstoff Delphinidin im Delphinidin-SBE-ß-CD in Kombination mit TRAIL zuzuschreiben. Delphinidin-SBE-ß-CD is in combination with TRAIL in accordance with the opposite delphinidin SBE-ß-CD alone increased apoptosis rates (Example 9) as well as the greater loss of cell viability (Example 10) a sy ¬ nergistische and greater blockage of cell proliferation (see Figure 14). A stronger block of cell proliferation can also be found in the combination of delphinidin Cl with TRAIL against TRAIL alone and delphinidin alone (see Figure 15). No enhancement of TRAIL sensitivity was observed with SBE-β-CD (see Figures 16 and 17), ie the antiproliferative enhancements described above are unambiguously attributable to the tested drug delphinidin in the delphinidin-SBE-β-CD in combination with TRAIL.
IV. Zytotoxizitätsuntersuchungen - Wirkung von Delphinidin und des Delphinidin-SBE-ß-CD Komplexes auf die Vitalität von Zellen
Beispiel 12 IV. Cytotoxicity Studies - Effect of delphinidin and the delphinidin-SBE-β-CD complex on the vitality of cells Example 12
(Wirkung von Delphinidin und des Delphinidin-SBE-ß-CD Komplexes auf die Vitalität von humanen Fibroblasten und Endo- thelzellen) (Effect of Delphinidin and the Delphinidin-SBE-β-CD Complex on the Vitality of Human Fibroblasts and Endothelial Cells)
Verwendete Zellen Used cells
Bei den verwendeten Zellen wurde auf primäre, selbst iso¬ lierte Fibroblasten und mikrovaskuläre Endothelzellen aus humaner Spenderhaut zurückgegriffen. In the cells used was to primary, even resorted iso ¬ profiled fibroblasts and microvascular endothelial cells from human donor skin.
Für die Isolierung der Fibroblasten wurde der Zellverband des Gewebes durch eine einstündige Inkubation mit Trypsin- EDTA-Lösung gelockert. Um die Ablösungsreaktion der Zellen zu beenden, wurde zur Haut Stoppmedium hinzu gegeben. Darauf wurde das Hautstück noch zweimal in PBS geschwenkt, so dass eine Fibroblastensuspension erhalten wurde. Die gewonnenen Fibroblasten wurden bei 4°C über 5 min mit 1000 Umdrehungen abzentrifugiert und nach Quantifizierung verwendet . For the isolation of the fibroblasts, the cell structure of the tissue was loosened by a one-hour incubation with trypsin-EDTA solution. To stop the detachment reaction of the cells, stop medium was added to the skin. Then the piece of skin was swung twice in PBS, so that a fibroblast suspension was obtained. The fibroblasts obtained were centrifuged off at 4 ° C. for 5 min at 1000 revolutions and used after quantification.
Die Gewinnung der humanen dermalen mikrovaskuläre Endothelzellen erfolgte mittels der dem Fachmann bekannten Standardmethode von Hewett and Murray (1993) [Hewett P.W. and Murray J.C. (1993) Immunomagnetic purification of human microvessel endothelial cells using Dynabeads coated monoc¬ lonal antibodies to PECAM-1. Eur J Cell Biol 62: 451-454; Hewett P.W. & Murray J.C. (1993) Human microvessel endothe¬ lial cells: Isolation, culture and characterization . In Vi¬ tro Cell Biol: 823-830].
Verwendete Messverfahren The recovery of human dermal microvascular endothelial cells was carried out using the known to the expert standard method of Hewett and Murray (1993) [Hewett PW and Murray JC (1993) Immunomagnetic purification of human microvessel endothelial cells using Dynabeads coated MONOC ¬ lonal antibodies to PECAM -1. Eur J Cell Biol 62: 451-454; Hewett PW & Murray JC (1993) Human microvessel endothe ¬ LiAl cells: isolation, culture and characterization. In Vi ¬ tro Cell Biol: 823-830]. Used measuring methods
In den Experimenten gemäß Beispiel 12 wurde die Wirkung der untersuchten Substanzen auf die Vitalität von humanen In the experiments according to Example 12, the effect of the substances tested on the vitality of human
Fibroblasten und Endothelzellen mit Hilfe des ATP- Lumineszenz-Assays untersucht, die dem Fachmann bekannt ist und nachfolgend der Vollständigkeit halber kurz zusammenge- fasst ist. Examined fibroblasts and endothelial cells using the ATP luminescence assay, which is known in the art and is briefly summarized below for the sake of completeness.
Nach dem Zelltod, beispielsweise aufgrund der Einwirkung eines zytotoxischen Wirkstoffs, nimmt der intrazellulare ATP-Gehalt durch ATPasen vermittelte Degradation im starken Maße ab. Bei Inhibierung der endogenen ATPasen kann der durch Lyse freigesetzte ATP-Gehalt folglich als Maß für die Anzahl vitaler Zellen verwendet werden. Für die Quantifizierung wird das freigesetzte ATP an eine von dem Enzym Lu- ciferase katalysierte Reaktion gekoppelt, bei der in Gegen¬ wart vom Mg2+ die ATP-abhängige Oxidation des Substrats Lu- ciferin zu Oxyluciferin, Kohlendioxid, AMP, anorganisches Phosphat unter Freisetzung von Licht stattfindet. Die Menge des mit dem ATP-Gehalt korrelierten freigesetzten Lichtes wird gemessen und lässt einen mittelbaren quantifizierbaren Rückschluss auf die Vitalität der untersuchten Zellen zu. Gemessen wird in RLU (Relative Light Units), einer eigen¬ ständigen Einheit, die die Menge an ATP in der Probe widerspiegelt. Kits und Geräte für die ATP-Bestimmung sind dem Fachmann bekannt, beispielsweise der ATP-Test-Kit der Fa. Biothema AB, Haning, Schweden, und das tragbare Luminometer Lumino® der Fa. STRATEC Biomedical Systems AG, Birkenfeld, Deutschland. Zur Bestimmung des intrazellulären ATP werden üblicherweise 50 μΐ der zu untersuchenden Probe in ein
Glasröhrchen pippettiert, dazu 50 μΐ des Extractant- Reagenzes B/S zur Zell-Lyse und 400 μΐ des ATP-HS-Reagenzes hinzugefügt und nach anschließender Mischung das verschlos¬ sene Röhrchen in das Luminometer zur automatischen Messung und Auswertung der Lichtemission gegeben. After cell death, for example, due to the action of a cytotoxic agent, the intracellular ATP content decreases strongly by ATPase-mediated degradation. Upon inhibition of the endogenous ATPases, the liberated ATP content can thus be used as a measure of the number of vital cells. For the quantification, the released ATP is coupled to a reaction catalyzed by the enzyme luciferase, in which, in the presence of Mg 2+, the ATP-dependent oxidation of the substrate luciferin to oxyluciferin, carbon dioxide, AMP, inorganic phosphate with release of light takes place. The amount of light released correlated with the ATP content is measured and allows an indirect quantifiable inference to the vitality of the examined cells. Is measured in RLU (Relative Light Units), an intrinsically ¬ permanent unit that reflects the amount of ATP in the sample. Kits and devices for ATP determination are known in the art, for example, the ATP test kit from. Biothema AB, Haning, Sweden, and the portable luminometer Lumino® Fa. STRATEC Biomedical Systems AG, Birkenfeld, Germany. To determine the intracellular ATP usually 50 .mu.ΐ of the sample to be examined in a Pippettiert glass tube, to 50 μΐ of Extractant- reagent B / S for cell lysis and 400 μΐ of the ATP-HS reagent added and added after subsequent mixing the verschlos ¬ sene tube in the luminometer for automatic measurement and evaluation of the light emission.
Im Vorfeld der Wirkstoffbehandlung und Anwendung des ATP- Lumineszenz-Assays wurden im vorliegendem Fall die humanen Fibroblasten- bzw. Endothelzellen mit Dulbecco ' s Modified Eagle's Medium (DMEM-Medium) auf 96-Well-Mikrotiterplatten ausgesät (5000 Zellen/Well) und für 3 Tage kultiviert. Nach dieser Anheftungs- und Wachstumsphase der Zellen im Brut¬ schrank fand die Substanzbehandlung mit ZeitIntervallen von Tag 1-Tag 4 (Endothelzellen) und 4h, 24h, 48h und 96h Prior to the drug treatment and application of the ATP luminescence assay in the present case, the human fibroblast or endothelial cells were seeded with Dulbecco's Modified Eagle's Medium (DMEM medium) on 96-well microtiter plates (5000 cells / well) and for Cultivated for 3 days. After this Anheftungs- and growth phase of the cells in the incubator cabinet ¬ substance treatment took place at time intervals from day 1-day 4 (endothelial cells) and 4h, 24h, 48h and 96h
(Fibroblasten) statt, wofür jeweils in 3 Wells in ΙΟΟμΙ DMEM-Medium gelöster Wirkstoff DelphinidinCl bzw. Delphini- din-SBE-ß-CD Komplex in unterschiedlichen Konzentrationen (0.1 μΜ; 3,2 μΜ; 100 μΜ) hinzugefügt wurde. Als Referenz¬ kontrolle wurden Zellen mit substanzfreiem Medium verwendet. Die Abnahme der Substanzen erfolgte nach 3 Stunden, gefolgt von einer Waschung mit Dulbecco ' s Phosphate Buffe- red Saline (PBS) . Die Endpunktmessung erfolgte mittels des bereits zu Beispiel 7 erwähnten WST-l-Assays . For this purpose, the active substance delphinidinCl or delphinidin-SBE-β-CD complex dissolved in 3 wells in ΙΟΟμΙ DMEM medium was added in different concentrations (0.1 μΜ, 3.2 μΜ, 100 μΜ). As a reference ¬ control cells were used with substance-free medium. The decrease of the substances took place after 3 hours, followed by a washing with Dulbecco's Phosphate Buffe red Saline (PBS). The end point measurement was carried out by means of the WST-1 assay already mentioned in Example 7.
Die Versuchsergebnisse des ATP-Lumineszenz-Assays sind in den Figuren 18 (Endothelzellen) und 19 (Fibroblasten) graphisch aufbereitet und lassen sich wie folgt zusammenfas¬ sen : The test results of the ATP luminescence assays are shown in Figures 18 (endothelial cells) and 19 (fibroblasts) graphically processed and can be as follows zusammenfas ¬ sen:
DelphinidinCl sowie Delphinidin-SBE-ß-CD zeigen bei den untersuchten Wirkstoffkonzentration (0,1-100 μΜ keine signifikanten zytotoxischen Effekte auf humane DelphinidinCl and delphinidin-SBE-ß-CD show no significant cytotoxic effects on human at the investigated drug concentration (0.1-100 μΜ)
Fibroblasten und Endothelzellen.
Damit erschließt sich eine völlig neue und besonders geeig¬ nete Wirkstoffgruppe zur Krebsbehandlung, weisen die untersuchten Wirkstoffe DelphinidinCl sowie Delphinidin-SBE-ß-CD doch einerseits die erwünschten antiproliferative Effekte auf Krebszellen (Melanomzellen) auf (Beispiele 6-11), vermeiden aber anderseits die bei therapeutischen Wirkstoff¬ konzentrationen in der Regel zu erwartenden unerwünschten zytotoxischen (Neben- ) Wirkungen auf Nicht-Krebszellen gemäß vorliegendem Beispiel 12.
Fibroblasts and endothelial cells. This opens up a completely new and particularly geeig ¬ designated agent group opens for cancer treatment, the tested drugs DelphinidinCl and delphinidin-SBE-.beta.-CD have but one hand the desired antiproliferative effects on cancer cells (melanoma cells) to (examples 6-11), but avoid the other hand, the expected therapeutic agent at concentrations ¬ usually undesirable cytotoxic (side) effects on non-cancer cells based on the present example 12. Fig.
Claims
Zusammensetzung umfassend Comprehensive composition
einen Komplex aus Delphinidin und einem Sulfoalky- lether-ß-Cyclodextrin zur Verwendung bei der Behandlung von malignem Melanom. a complex of delphinidin and a sulfoalkyl ether-ß-cyclodextrin for use in the treatment of malignant melanoma.
Zusammensetzung zur Verwendung nach Anspruch 1, dadurch gekennzeichnet, dass das Sulfoalkylether-ß-Cyclodextrin im Komplex ein Sulfobutylether-ß-Cyclodextrin ist. Composition for use according to claim 1, characterized in that the sulfoalkyl ether-ß-cyclodextrin in the complex is a sulfobutyl ether-ß-cyclodextrin.
Zusammensetzung zur Verwendung nach einem der vorangehenden Ansprüche, dadurch gekennzeichnet, dass im Kom¬ plex der Substitutionsgrad des Cyclodextrins mit Sulfo- alkylethergruppen 3 bis 8, vorzugsweise 4 bis 8, weiter vorzugsweise 5 bis 8, weiter vorzugsweise 6 bis 7 be¬ trägt . Composition for use according to one of the preceding claims, characterized in that in the complex the degree of substitution of the cyclodextrin with sulfoalkyl ether groups is 3 to 8, preferably 4 to 8, more preferably 5 to 8, more preferably 6 to 7.
Zusammensetzung zur Verwendung nach einem der vorangehenden Ansprüche, dadurch gekennzeichnet, dass die Zu¬ sammensetzung eine therapeutisch wirksame Menge an dem Komplex aus Delphinidin und Sulfoalkylether-ß- Cyclodextrin umfasst. Composition for use according to one of the preceding claims, characterized in that the composition comprises a therapeutically effective amount of the complex of delphinidin and sulfoalkyl ether-ß-cyclodextrin.
Zusammensetzung zur Verwendung nach einem der vorangehenden Ansprüche, dadurch gekennzeichnet, dass die Zu¬ sammensetzung als Monopräparat verwendet wird. Composition for use according to one of the preceding claims, characterized in that the composition is used as a monopreparation.
Zusammensetzung zur Verwendung nach einem der Ansprüche, dadurch gekennzeichnet, dass die Zusammensetzung
mindestens eine weitere therapeutisch aktive Substanz umfasst . Composition for use according to one of the claims, characterized in that the composition comprises at least one further therapeutically active substance.
7. Zusammensetzung zur Verwendung nach Anspruch 6, dadurch gekennzeichnet, dass die therapeutisch aktive Substanz ein Tumornekrosefaktor-verwandter Apoptose-induzierter Ligand (Tumor Necrosis Factor Related Apoptosis Indu- cing Ligand - TRAIL) ist. 7. Composition for use according to claim 6, characterized in that the therapeutically active substance is a tumor necrosis factor-related apoptosis-induced ligand (Tumor Necrosis Factor Related Apoptosis Inducing Ligand - TRAIL).
8. Zusammensetzung zur Verwendung nach Anspruch 6, dadurch gekennzeichnet, dass die therapeutisch aktive Substanz ausgewählt ist aus der Gruppe bestehend aus 8. Composition for use according to claim 6, characterized in that the therapeutically active substance is selected from the group consisting of
Zytostatika, cytostatics,
Interferone, vorzugsweise Alpha- und/oder Beta- Interferone, weiter vorzugsweise Interferon alpha 2-a und/oder alpha 2-b, und Interferons, preferably alpha and/or beta interferons, more preferably interferon alpha 2-a and/or alpha 2-b, and
- Tumorvakzine. - Tumor vaccines.
9. Zusammensetzung zur Verwendung nach einem der vorangehenden Ansprüche, weiter umfassend ein oder mehrere pharmazeutische Hilfs- und/oder Zusatzstoffe, vorzugs¬ weise ausgewählt aus der Gruppe bestehend aus einen pharmazeutisch akzeptablen Träger, Füllstoffe, Geruchsstoffe und Stabilisatoren. 9. Composition for use according to one of the preceding claims, further comprising one or more pharmaceutical auxiliaries and / or additives, preferably selected from the group consisting of a pharmaceutically acceptable carrier, fillers, odorants and stabilizers.
10. Zusammensetzung zur Verwendung nach einem der vorangehenden Ansprüche in einer Formulierungsform zur Verabreichung in einer Form ausgewählt aus der Gruppe beste¬ hend aus oral, rektal, parenteral, einschließlich intraperitoneal, perkutan, subkutan, intramuskulär, intravenös, ophthalmisch, pulmonal und nasal.
10. Composition for use according to one of the preceding claims in a formulation form for administration in a form selected from the group consisting of oral, rectal, parenteral, including intraperitoneal, percutaneous, subcutaneous, intramuscular, intravenous, ophthalmic, pulmonary and nasal.
11. Zusammensetzung zur Verwendung nach Anspruch 10, dadurch gekennzeichnet, dass die Verabreichungsform aus¬ gewählt ist aus der Gruppe bestehend aus Tablette, Kap¬ sel, Suspension, Aerosol, Lösung, Creme, Paste, Lotion, Gel und Salbe. 11. Composition for use according to claim 10, characterized in that the administration form is selected from the group consisting of tablet, capsule , suspension, aerosol, solution, cream, paste, lotion, gel and ointment.
12. Zusammensetzung zur Verwendung nach einem der vorangehenden Ansprüche, dadurch gekennzeichnet, dass der Kom¬ plex aus Delphinidin und dem Sulfoalkylether—ß- Cyclodextrin in einer galenischen Zubereitung zur kontrollierten und/oder zeitverzögerten Freisetzung des Delphinidins eingesetzt wird. 12. Composition for use according to one of the preceding claims, characterized in that the complex of delphinidin and the sulfoalkyl ether-ß-cyclodextrin is used in a galenic preparation for the controlled and / or time-delayed release of the delphinidin.
13. Zusammensetzung zur Verwendung nach einem der vorangehenden Ansprüche, dadurch gekennzeichnet, dass die Be¬ handlung des malignem Melanoms bei einem Objekt erfolgt, das einer Behandlung ausgewählt aus der Gruppe bestehend aus 13. Composition for use according to one of the preceding claims, characterized in that the treatment of the malignant melanoma takes place in an object which is subject to a treatment selected from the group consisting of
- chirurgischer Entfernung von Melanomzellen oder von mit malignem Melanom befallenen Gewebe, - surgical removal of melanoma cells or tissue affected by malignant melanoma,
- Strahlentherapie, - radiation therapy,
- Immuntherapie, - immunotherapy,
- Chemotherapie und - chemotherapy and
- Interferon-Behandlung, - interferon treatment,
unterzogen wurde, unterzogen wird oder darauf vorbereitet wird.
has undergone, is undergoing or is being prepared for.
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201380064588.0A CN104918622A (en) | 2012-12-11 | 2013-11-28 | Delphinidin for combating melanoma cells |
| CA2893881A CA2893881C (en) | 2012-12-11 | 2013-11-28 | Delphinidin for combating melanoma cells |
| KR1020157018731A KR102164174B1 (en) | 2012-12-11 | 2013-11-28 | Delphinidin for combating melanoma cells |
| US14/651,262 US9949947B2 (en) | 2012-12-11 | 2013-11-28 | Delphinidin for combating melanoma cells |
| EP13815384.6A EP2931287B1 (en) | 2012-12-11 | 2013-11-28 | Delphinidin for combating melanoma cells |
| JP2015546927A JP6234475B2 (en) | 2012-12-11 | 2013-11-28 | Delphinidin to combat melanoma cells |
| HK16103397.5A HK1216229A1 (en) | 2012-12-11 | 2016-03-23 | Delphinidin for combating melanoma cells |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE102012222777 | 2012-12-11 | ||
| DE102012222777.6 | 2012-12-11 | ||
| EP13150909.3 | 2013-01-11 | ||
| EP13150909 | 2013-01-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2014090583A1 true WO2014090583A1 (en) | 2014-06-19 |
Family
ID=49679524
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/EP2013/074957 WO2014090583A1 (en) | 2012-12-11 | 2013-11-28 | Delphinidin for combating melanoma cells |
| PCT/EP2013/074991 WO2014090586A1 (en) | 2012-12-11 | 2013-11-28 | Delphinidin for combating melanoma cells |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/EP2013/074991 WO2014090586A1 (en) | 2012-12-11 | 2013-11-28 | Delphinidin for combating melanoma cells |
Country Status (8)
| Country | Link |
|---|---|
| US (2) | US9511047B2 (en) |
| EP (2) | EP2931287B1 (en) |
| JP (2) | JP6234475B2 (en) |
| KR (2) | KR102164174B1 (en) |
| CN (2) | CN104936601A (en) |
| CA (2) | CA2893883A1 (en) |
| HK (2) | HK1215392A1 (en) |
| WO (2) | WO2014090583A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9925274B2 (en) | 2012-11-15 | 2018-03-27 | Sapiotec Gmbh | Delphinidin complex as an antiphlogistic or immunosuppressive active ingredient |
| US9949947B2 (en) | 2012-12-11 | 2018-04-24 | Sapiotec Gmbh | Delphinidin for combating melanoma cells |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5134127A (en) | 1990-01-23 | 1992-07-28 | University Of Kansas | Derivatives of cyclodextrins exhibiting enhanced aqueous solubility and the use thereof |
| US20050013880A1 (en) * | 2003-03-06 | 2005-01-20 | Magnuson Bernadene Ann | Anthocyanin-rich compositions and methods for inhibiting cancer cell growth |
| WO2006076387A2 (en) * | 2005-01-11 | 2006-07-20 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention | Cyanidin-3-glucoside as an anti-neoplastic agent |
| US20090270348A1 (en) * | 2008-04-28 | 2009-10-29 | Antle Vincent | Sulfoalkyl Ether Cyclodextrin Compositions |
Family Cites Families (48)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5946228A (en) | 1982-09-08 | 1984-03-15 | Zeria Shinyaku Kogyo Kk | Preparation of water-soluble and lymph-transitional drug containing biologically active organic compound |
| HU199444B (en) | 1985-09-10 | 1990-02-28 | Chinoin Gyogyszer Es Vegyeszet | Process for producing 7-isopropoxy-isoflavone-cyclodextrene inclusion complex and pharmaceutical compositions therefrom |
| GB8907008D0 (en) | 1989-03-28 | 1989-05-10 | Inverni Della Beffa Spa | Intermediates useful for the synthesis for delphinidin chloride |
| US5376645A (en) * | 1990-01-23 | 1994-12-27 | University Of Kansas | Derivatives of cyclodextrins exhibiting enhanced aqueous solubility and the use thereof |
| ZA962214B (en) | 1995-04-10 | 1996-10-07 | Farmarc Nederland Bv | Pharmaceutical composition |
| US5763223A (en) * | 1995-06-29 | 1998-06-09 | Immunex Corporation | DNA encoding a cytokine that induces apoptosis |
| GB2307176A (en) | 1995-11-15 | 1997-05-21 | Todd Selwyn Everest | Anti-inflammatory clathrating agents for topical use |
| HUP9701945A3 (en) | 1997-11-10 | 2000-04-28 | Hexal Ag | Pharmaceutical composition for injection containing cyclodextrin and taxoids |
| JP4101346B2 (en) | 1998-02-18 | 2008-06-18 | ダイセル化学工業株式会社 | Oxidation catalyst system and oxidation method using the same |
| AU759280C (en) | 1998-02-23 | 2004-01-22 | Cyclops, Ehf | High-energy cyclodextrin complexes |
| US6699849B1 (en) | 1998-02-23 | 2004-03-02 | Cyclops, Ehf. | Cyclodextrin complexes of benzodiazepines |
| GB2350297A (en) | 1999-05-27 | 2000-11-29 | Abbott Lab | Injectable halogenated anesthetic formulation in emulsion form |
| AU6527300A (en) | 1999-08-11 | 2001-03-05 | Josef Pitha | Potentiation of inclusion complex formation of cyclodextrin derivatives |
| AU7937500A (en) | 1999-10-27 | 2001-05-08 | Farmarc Nederland Bv | Pharmaceutical composition containing midazolam |
| JPWO2002022847A1 (en) * | 2000-09-12 | 2004-01-22 | 明治製菓株式会社 | Process for producing purified anthocyanins and crystalline anthocyanins |
| JP4334229B2 (en) | 2001-03-20 | 2009-09-30 | サイデックス・ファーマシューティカルズ・インコーポレイテッド | Formulation containing propofol and sulfoalkyl ether cyclodextrin |
| US7034013B2 (en) | 2001-03-20 | 2006-04-25 | Cydex, Inc. | Formulations containing propofol and a sulfoalkyl ether cyclodextrin |
| JP2003171274A (en) | 2001-12-07 | 2003-06-17 | Tomihiko Higuchi | Medicinal composition and disinfectant for treating infection with drug-resistant microorganism |
| EP1469886B8 (en) | 2002-02-01 | 2008-01-09 | Shimoda Biotech (PTY) LTD | Lyophilized pharmaceutical composition of propofol |
| JP2004238336A (en) * | 2003-02-07 | 2004-08-26 | Sanei Gen Ffi Inc | Method for producing easily water-soluble inclusion flavonoids |
| MXPA05010330A (en) | 2003-03-28 | 2006-05-31 | Ivax Corp | Cladribine formulations for improved oral and transmucosal delivery. |
| JPWO2005067905A1 (en) | 2004-01-14 | 2007-12-27 | 株式会社大塚製薬工場 | Propofol-containing fat emulsion |
| CN1317961C (en) | 2004-03-25 | 2007-05-30 | 吴朝琴 | Strawberry freeze-drying process |
| CN100342026C (en) * | 2004-05-13 | 2007-10-10 | 深圳新鹏生物工程有限公司 | Method for preparing recombined apoptosis induction ligand relevant to human soluble tumor necrosis factor |
| DE102005017775A1 (en) | 2005-04-13 | 2006-10-19 | Schering Ag | New complex of vitamin-D-compounds with 5Z,7E,10(19)-triene system and methylene derivatives of beta-cyclodextrin, useful for the preparation of medicament and to treat psoriasis |
| KR100733913B1 (en) * | 2005-08-30 | 2007-07-02 | 한국화학연구원 | Agent for the protection of brain cell and Agent for the prevention or treatment of cerebral infarction and stroke comprising anthocyanin |
| US7645748B2 (en) | 2006-04-03 | 2010-01-12 | Forbes Medi-Tech Inc. | Sterol/stanol phosphorylnitroderivatives and use thereof |
| US9000048B2 (en) | 2006-11-28 | 2015-04-07 | Wisconsin Alumni Research Foundation | Fluoropolymer-based emulsions for the intravenous delivery of fluorinated volatile anesthetics |
| KR100756656B1 (en) | 2006-12-12 | 2007-09-10 | 한국화학연구원 | Health food for the prevention and improvement of cerebral infarction or stroke comprising anthocyanin |
| KR100880876B1 (en) | 2007-04-11 | 2009-01-30 | 경상대학교산학협력단 | Pharmaceutical composition for the prevention and treatment of the ischemia-reperfusion injury in the flaps or tissues containing anthocyanin extracted from the Black soybean seed coat |
| WO2009018326A2 (en) * | 2007-07-31 | 2009-02-05 | Limerick Biopharma, Inc. | Soluble pyrone analogs methods and compositions |
| JP5574600B2 (en) | 2007-12-17 | 2014-08-20 | 日本製薬工業株式会社 | Preventive and therapeutic agent for resorbable bone disease |
| EP2106786A1 (en) | 2008-04-04 | 2009-10-07 | Roewer, Norbert, Univ.-Prof. Dr. med. | Pharmaceutical preparation comprising permethylated cyclodextrin |
| WO2010110328A1 (en) | 2009-03-25 | 2010-09-30 | 三栄源エフ・エフ・アイ株式会社 | Readily water-soluble isoquercitrin composition |
| US9352015B2 (en) | 2009-06-05 | 2016-05-31 | The Regents Of The University Of Colorado, A Body Corporate | Antimicrobial peptides |
| WO2011048479A2 (en) | 2009-10-21 | 2011-04-28 | Maqui New Life S.A. | Compositions that include anthocyanidins and methods of use |
| EP2345427A1 (en) | 2010-01-14 | 2011-07-20 | SapioTec GmbH | Fluoran complex |
| RU2552927C2 (en) | 2010-03-13 | 2015-06-10 | Истпонд Лабораториз Лимитед | Fat-biding composition |
| DE102010042615A1 (en) | 2010-10-19 | 2012-04-19 | Wacker Chemie Ag | 1: 1, 2: 1 or 3: 1 complex consisting of a cyclodextrin or cyclodextrin derivative and a halogenated ether, its preparation and its use as a sleep aid |
| EP4043037A1 (en) | 2010-12-31 | 2022-08-17 | Eastpond Laboratories Limited | Cellular hydration compositions containing cyclodextrins |
| DK2484350T3 (en) | 2011-02-04 | 2016-08-01 | Norbert Univ -Prof Dr Med Roewer | A pharmaceutical composition containing a complex of a cyclodextrin with a propofolsalt |
| CA2869056C (en) | 2012-03-30 | 2020-02-25 | Sapiotec Gmbh | Anthocyanidin complex |
| CN104302183B (en) | 2012-03-30 | 2017-03-01 | 赛博尔泰克股份公司 | The application to anti-Staphylococcus aureus for the delphinidin |
| CN104780925A (en) * | 2012-10-17 | 2015-07-15 | 赛博尔泰克股份公司 | Anthocyanidin complex for the treatment of multiple myeloma |
| KR102147084B1 (en) | 2012-11-15 | 2020-08-24 | 자피오텍 게엠베하 | Delphinidin complex as an antiphlogistic or immunosuppressive active ingredient |
| EP2931287B1 (en) | 2012-12-11 | 2017-10-04 | Sapiotec GmbH | Delphinidin for combating melanoma cells |
| EP2913366A1 (en) | 2014-02-28 | 2015-09-02 | SapioTec GmbH | Anthocyanidin complex |
| EP2913050A1 (en) | 2014-02-28 | 2015-09-02 | SapioTec GmbH | Method for producing a flurane complex |
-
2013
- 2013-11-28 EP EP13815384.6A patent/EP2931287B1/en not_active Not-in-force
- 2013-11-28 WO PCT/EP2013/074957 patent/WO2014090583A1/en active Application Filing
- 2013-11-28 EP EP13798323.5A patent/EP2931286B1/en not_active Not-in-force
- 2013-11-28 KR KR1020157018731A patent/KR102164174B1/en active IP Right Grant
- 2013-11-28 CN CN201380064614.XA patent/CN104936601A/en active Pending
- 2013-11-28 US US14/651,264 patent/US9511047B2/en not_active Expired - Fee Related
- 2013-11-28 CA CA2893883A patent/CA2893883A1/en not_active Abandoned
- 2013-11-28 CA CA2893881A patent/CA2893881C/en not_active Expired - Fee Related
- 2013-11-28 JP JP2015546927A patent/JP6234475B2/en not_active Expired - Fee Related
- 2013-11-28 JP JP2015546930A patent/JP6234476B2/en not_active Expired - Fee Related
- 2013-11-28 US US14/651,262 patent/US9949947B2/en not_active Expired - Fee Related
- 2013-11-28 CN CN201380064588.0A patent/CN104918622A/en active Pending
- 2013-11-28 WO PCT/EP2013/074991 patent/WO2014090586A1/en active Application Filing
- 2013-11-28 KR KR1020157018732A patent/KR20150107742A/en not_active Application Discontinuation
-
2016
- 2016-03-23 HK HK16103394.8A patent/HK1215392A1/en unknown
- 2016-03-23 HK HK16103397.5A patent/HK1216229A1/en not_active IP Right Cessation
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5134127A (en) | 1990-01-23 | 1992-07-28 | University Of Kansas | Derivatives of cyclodextrins exhibiting enhanced aqueous solubility and the use thereof |
| US20050013880A1 (en) * | 2003-03-06 | 2005-01-20 | Magnuson Bernadene Ann | Anthocyanin-rich compositions and methods for inhibiting cancer cell growth |
| WO2006076387A2 (en) * | 2005-01-11 | 2006-07-20 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention | Cyanidin-3-glucoside as an anti-neoplastic agent |
| US20090270348A1 (en) * | 2008-04-28 | 2009-10-29 | Antle Vincent | Sulfoalkyl Ether Cyclodextrin Compositions |
| WO2009134347A2 (en) | 2008-04-28 | 2009-11-05 | Cydex Pharmaceuticals, Inc. | Sulfoalkyl ether cyclodextrin compositions |
Non-Patent Citations (14)
| Title |
|---|
| "Remington's Pharmaceutical Sciences", MACK PUBLISHING CO |
| AFAQ FARRUKH ET AL: "Delphinidin, an anthocyanidin in pigmented fruits and vegetables, protects human HaCaT keratinocytes and mouse skin against UVB-mediated oxidative stress and apoptosis", JOURNAL OF INVESTIGATIVE DERMATOLOGY, vol. 127, no. 1, January 2007 (2007-01-01), pages 222 - 232, XP002692596, ISSN: 0022-202X * |
| BIOLINK: "Biolink synthetic anthocyanin molecules becomes cancer figthers", 22 May 2009 (2009-05-22), pages 1 - 4, XP002692595, Retrieved from the Internet <URL:http://www.biolink.no/biolink/cancer-article95-6.html> [retrieved on 20130221] * |
| BRUGGEN J.; SORG C.: "Detection of phenotypic differences on human malignant melanoma lines and their variant sublines with monoclonal antibodies", CANCER IMMUNOL IMMUNOTHER, vol. 15, 1983, pages 200 - 205 |
| DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; September 2012 (2012-09-01), VOERSMANN H ET AL: "Three dimensional melanoma treatment using the death ligand TRAIL", XP009176203, Database accession no. PREV201200611221 * |
| HEWETT P.W.; MURRAY J.C.: "Human microvessel endothelial cells: Isolation, culture and characterization", IN VITRO CELL BIOL, 1993, pages 823 - 830 |
| HEWETT P.W.; MURRAY J.C.: "Immunomagnetic purification of human microvessel endothelial cells using Dynabeads coated monoclonal antibodies to PECAM-1", EUR J CELL BIOL, vol. 62, 1993, pages 451 - 454, XP009112370 |
| JOURNAL OF INVESTIGATIVE DERMATOLOGY, vol. 132, no. Suppl. 2, September 2012 (2012-09-01), 42ND ANNUAL MEETING OF THE EUROPEAN-SOCIETY-FOR-DERMATOLOGICAL-RESEARCH (ESDR); VENICE, ITALY; SEPTEMBER 07 -10, 2012, pages S129, ISSN: 0022-202X(print) * |
| PLÖTZ ET AL.: "Mutual Regulation of Bcl-2 Proteins Independent of the BH3 Domain as Shown by the BH3-Lacking Protein Bcl-xAK", PLOS ONE, vol. 7, no. 4, 2012, pages E34549 |
| RICCARDI C.; NICOLETTI I.: "Analysis of apoptosis by propidium iodide staining and flow cytometry", NAT. PROTOC, vol. 1, 2006, pages 1458 - 1461, XP055130538, DOI: doi:10.1038/nprot.2006.238 |
| SERAFINO ANNALUCIA ET AL: "Differentiation of human melanoma cells induced by cyanidin-3-O-beta-glucopyranoside", FASEB JOURNAL, vol. 18, no. 12, September 2004 (2004-09-01), pages 1 - 25, XP002692597, ISSN: 0892-6638 * |
| UEDA ET AL.: "Evaluation of a Sulfobutyl Ether ß-Cyclodextrin as a Solubizing/Stabilizing Agent for Several Drugs", DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY, vol. 24, no. 9, 1998, pages 863 - 867, XP009040590, DOI: doi:10.3109/03639049809088532 |
| UEDA H ET AL: "EVALUATION OF A SULFOBUTYL ETHER BETA-CYCLODEXTRIN AS A SOLUBILIZING/STABILIZING AGENT FOR SEVERAL DRUGS", DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY, NEW YORK, NY, US, vol. 24, no. 9, 1 September 1998 (1998-09-01), pages 863 - 867, XP009040590, ISSN: 0363-9045, DOI: 10.3109/03639049809088532 * |
| WANG L S ET AL: "Anthocyanins and their role in cancer prevention", CANCER LETTERS, NEW YORK, NY, US, vol. 269, no. 2, 8 October 2008 (2008-10-08), pages 281 - 290, XP025496527, ISSN: 0304-3835, [retrieved on 20080620], DOI: 10.1016/J.CANLET.2008.05.020 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9925274B2 (en) | 2012-11-15 | 2018-03-27 | Sapiotec Gmbh | Delphinidin complex as an antiphlogistic or immunosuppressive active ingredient |
| US9949947B2 (en) | 2012-12-11 | 2018-04-24 | Sapiotec Gmbh | Delphinidin for combating melanoma cells |
Also Published As
| Publication number | Publication date |
|---|---|
| HK1216229A1 (en) | 2016-10-28 |
| CA2893883A1 (en) | 2014-06-19 |
| HK1215392A1 (en) | 2016-08-26 |
| KR20150107741A (en) | 2015-09-23 |
| CA2893881C (en) | 2020-09-22 |
| US9511047B2 (en) | 2016-12-06 |
| US20150328336A1 (en) | 2015-11-19 |
| CN104936601A (en) | 2015-09-23 |
| EP2931286A1 (en) | 2015-10-21 |
| US20150320719A1 (en) | 2015-11-12 |
| JP2016502985A (en) | 2016-02-01 |
| CN104918622A (en) | 2015-09-16 |
| KR20150107742A (en) | 2015-09-23 |
| CA2893881A1 (en) | 2014-06-19 |
| KR102164174B1 (en) | 2020-10-12 |
| EP2931286B1 (en) | 2017-10-04 |
| EP2931287B1 (en) | 2017-10-04 |
| JP6234475B2 (en) | 2017-11-22 |
| US9949947B2 (en) | 2018-04-24 |
| EP2931287A1 (en) | 2015-10-21 |
| JP6234476B2 (en) | 2017-11-22 |
| JP2016502984A (en) | 2016-02-01 |
| WO2014090586A1 (en) | 2014-06-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fan et al. | Inhibition of autophagy overcomes the nanotoxicity elicited by cadmium-based quantum dots | |
| DE69430126T2 (en) | CYCLODEXTRINE FOR REDUCING THE VESSEL LEAKAGE CAUSED BY CYTOTOXIC SUBSTANCES | |
| Tan et al. | The performance of doxorubicin encapsulated in chitosan–dextran sulphate microparticles in an osteosarcoma model | |
| Park et al. | Targeting and synergistic action of an antifungal peptide in an antibiotic drug-delivery system | |
| Botelho et al. | Hydroalcoholic extract from Nerium oleander L.(Apocynaceae) elicits arrhythmogenic activity | |
| Marchianti et al. | Gel formulations of Merremia mammosa (Lour.) accelerated wound healing of the wound in diabetic rats | |
| DE68910113T2 (en) | GROWTH INHIBITANT AND ITS USE. | |
| EP2919791B1 (en) | Delphinidincomplex as antiphlogistic or immunosuppressive agent | |
| EP2931286B1 (en) | Delphinidin for combating melanoma cells | |
| Tohamy et al. | Lucrative antioxidant effect of metformin against cyclophosphamide induced nephrotoxicity | |
| WO2014060548A1 (en) | Anthocyanidin complex for the treatment of multiple myeloma | |
| WO2013144306A1 (en) | Use of delphinidin against staphylococcus aureus | |
| DE102015210224A1 (en) | NEW ACTIVE FOR THE TREATMENT OF CANCER | |
| DE3510795A1 (en) | MICROBIAL EXTRACT (JOMOL), ITS PRODUCTION AND USE AS A DIAGNOSTIC AND MEDICINAL PRODUCT. DERIVATIVES OF JOMOL | |
| EP0173262A2 (en) | Use of 1,4-naphthoquinone derivatives in a low concentration as immunostimulating agents | |
| DE69433472T2 (en) | Recognin VACCINES | |
| Anjana et al. | Invitro Antioxidant Activity Of Fresh Aloe Vera Gel Against Oral Mucosal Lesions-An Invitro Study | |
| Chen et al. | Ultrasmall Ag6Cu2 clusters for accelerating wound healing through the activation of antibacterial activity and endogenous anti-inflammatory property | |
| WO1986000524A1 (en) | Oxindole alkaloids with properties stimulating the immunity system and preparations containing them | |
| DE102005056194A1 (en) | New lipopeptide compositions | |
| Kulikov et al. | Phototoxicity in vitro and safety in vivo of the emulsion photosensitizer based on furanocoumarins of Heracleum sosnowskyi | |
| EP1893242A2 (en) | Method for producing albumin conjugates comprising an x-ray contrast medium as the active substance | |
| DE102009035586A1 (en) | Intravesical instillation solutions for the treatment of bladder cancer | |
| KR20190094671A (en) | novel macromolecular compound comprising UDCA and use thereof | |
| Chauhan et al. | Pharmacokinetic Study of Aloin Nanoparticulate: Enhanced Oral Formulation Bioavailability |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 13815384 Country of ref document: EP Kind code of ref document: A1 |
|
| ENP | Entry into the national phase |
Ref document number: 2893881 Country of ref document: CA |
|
| ENP | Entry into the national phase |
Ref document number: 2015546927 Country of ref document: JP Kind code of ref document: A |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 14651262 Country of ref document: US |
|
| REEP | Request for entry into the european phase |
Ref document number: 2013815384 Country of ref document: EP |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 2013815384 Country of ref document: EP |
|
| ENP | Entry into the national phase |
Ref document number: 20157018731 Country of ref document: KR Kind code of ref document: A |